Nucleic acid detection

ABSTRACT

This invention discloses methods, compositions and kits for the detection of extremely low levels of nucleic acid, cells and cellular material in biological samples. The nucleic acid detection systems utilize either the pyrophosphorolysis reaction catalyzed by various polymerases or nuclease digestion coupled with pyrophosphorylation catalyzed by phosphoribosylpyrophosphate synthetase to produce either deoxyribonucleoside triphosphates or ribonucleoside triphosphates. dNTPs are transformed to ATP by the action of nucleoside diphosphate kinase. The ATP produced by these reactions may be detected by luciferase or NADH based detection systems. If more sensitive detection is required, schemes for the amplification of NTPs and dNTPS are provided. A detection system for cells or cellular material in a sample is provided wherein AMP and a high energy phosphate donor added to a sample are converted to ATP by the action of endogenous enzymes, followed by detection of the ATP.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, and inparticular to the detection of nucleic acids and cells. Methods andcompositions for detection of extremely low amounts of nucleic acids andcellular materials with ATP based detection systems are described.

BACKGROUND

Methods for producing large amounts of recombinant protein are wellknown. As the recombinant protein industry has developed, the need forvarious quality control assays has arisen. An example is the need forthe quantitation of nucleic acids present in recombinant proteinpreparations. Current guidelines require that the amount of nucleic acidpresent in recombinant therapeutic proteins be less than 10 pg of DNAper daily dose of recombinant protein. Therefore, methods for detectingextremely low amounts of nucleic acids are needed. Such methods wouldalso find widespread use for the quantitation of DNA in forensicsamples.

Several methods of detecting low levels of nucleic acid have beendescribed. The first method is based on classical hybridizationtechniques. This method utilizes radio-labeled nucleic acid probes whichbind to the DNA of interest. However, this method has severaldisadvantages including poor reproducibility, generation of largeamounts of waste reagent, and high background levels caused bynonspecific binding. Furthermore, this technique is generallyinappropriate for determining the presence of low amounts of DNA ofunknown sequence.

A second method of detecting nucleic acid utilizes fluorescent dyescapable of intercalating into nucleic acids. However, many interferingsubstances such as detergents, proteins, and lipids affect thereproducibility of the signal generated by this method.

A third method of detecting low levels of DNA utilizes biotinylatedsingle-stranded DNA binding protein (SSB), streptavidin, an anti-DNAantibody fused to urease, and biotinylated nitrocellulose as reagents.This assay is commercially available from Molecular Devices anddescribed in Kung et al., Picogram Quantitation of Total DNA UsingSNA-Binding Proteins in a Silicon Sensor-Based System, Anal. Biochem.187: 220-27 (1990). The assay is performed by incubating thestreptavidin, biotin-SSB, and the anti-DNA antibody together, allowing acomplex to be formed. The complex is then captured on the biotinylatedfilter, washed, and the amount of captured urease is read. This methodis highly sensitive but has several disadvantages. These disadvantagesinclude costly reagents and the need for extensive controls.

A fourth method involves the depolymerization or degradation of nucleicacids and detection of ATP by luciferase. Polynucleotide polymerases areresponsible for the synthesis of nucleic acids in cells. These enzymesare also capable of catalyzing other reactions as described in Deutscherand Kornberg, Enzymatic Synthesis of Deoxyribonucleic Acid, J. Biol.Chem. 244 (11):3019-28 (1969). Many, but not all, polymerases are ableto depolymerize nucleic acid in the presence of either phosphate orpyrophosphate.

U.S. Pat. No. 4,735,897 describes a method of detecting polyadenylatedmessenger RNA (poly(A)-mRNA). Depolymerization of poly(A)-mRNA in thepresence of phosphate has been shown to result in the formation of ADP,which can be converted by pyruvate kinase or creatine phosphokinase intoATP. RNA may also be digested by a ribonuclease to AMP, converted to ADPby adenylate kinase, and then converted to ATP by pyruvate kinase.

The ATP so produced is detected by a luciferase detection system. In thepresence of ATP and 02, luciferase catalyzes the oxidation of luciferin,producing light which can then be quantitated using a luminometer.Additional products of the reaction are AMP, pyrophosphate andoxyluciferin.

The presence of ATP-generating enzymes in all organisms also allows theuse of a luciferase system for detecting the presence or amounts ofcontaminating cells in a sample, as described in U.S. Pat. No.5,648,232. For example, ADP may be added to a sample suspected ofcontaining contaminating cells. The ADP is converted by enzymes of thecell into ATP which is detected by a luciferase assay, as describedabove. The disadvantage of this method is the relative instability ofthe ADP substrate.

What is needed in the art are reliable, cost-effective methods ofdetecting extremely low levels of nucleic acids, cells, and cellularmaterial in a wide variety of samples. The present invention disclosesnovel methods for detecting low quantities of DNA, RNA and of cells.These methods take advantage of novel combinations of pyrophosphorolysisor enzymatic degradation of nucleic acids, conversion of dNTPs to ATP,the conversion of AMP directly to ATP, amplification of ATP to increasesensitivity, depolymerization of oligonucleotide probes, and optimizedreaction conditions.

SUMMARY OF THE INVENTION

A need exists for quality control assays for proteins produced byrecombinant methods. Current guidelines suggest that preparations ofrecombinant protein should contain less than 10 picograms of nucleicacid. There is also a need to be able to quantitate extremely low levelsof nucleic acids in forensics samples. Therefore, it is an object of thepresent invention to provide methods for detecting low amounts ofnucleic acids and low numbers of cells or cellular material. It is alsoan object of the invention to provide compositions for the detection ofnucleic acids and kits for the detection of nucleic acids.

In one embodiment of the present invention a method is provided fordetecting and/or assaying deoxyribonucleic acid in a reaction containingphosphate, adenosine 5′-diphosphate, or a combination thereof. Themethod comprises depolymerizing the nucleic acid at a terminalnucleotide by enzymatically cleaving the terminal internucleosidephosphodiester bond and reforming same with a pyrophosphate molecule toform a deoxyribonucleoside triphosphate molecule according to thefollowing reaction:

NA_(n)+PP_(i)→NA_(n−1)+dNTP

catalyzed by a DNA polymerase or reverse transcriptase selected from thegroup consisting of T4 polymerase, Taq polymerase, AMV reversetranscriptase, and MMLV reverse transcriptase. In a quantitative assayfor nucleic acids, the depolymerizing step is repeated essentially tocompletion or equilibrium to obtain at least two nucleoside triphosphatemolecules from a strand of minimally three nucleotides. For detection ofDNA, the depolymerizing step need not be repeated if there aresufficient nucleic acid molecules present to generate a signal. The nextstep involves enzymatically transferring terminal 5′ phosphate groupsfrom the deoxyribonucleoside triphosphate molecules to an adenosine5′-diphosphate molecule to form adenosine 5′-triphosphate according tothe following reaction:

dNTP*+ADP→NDP+ATP*

catalyzed by nucleoside diphosphate kinase and wherein P* is theterminal 5′ phosphate so transferred. The final step is the detection ofthe ATP, either by a luciferase detection system or NADH detectionsystem. The depolymerizing step and phosphate transferring step mayoptionally be performed in a single pot reaction. If greater sensitivityis desired, the ATP molecules produced by the phosphate transferringstep or the NTPs produced by the depolymerizing step may be amplified toform a plurality of ATP molecules.

In another embodiment of the present invention, a method is provided fordetecting polyadenylated mRNA in a reaction containing pyrophosphate.The polyadenylated mRNA is first depolymerized at a terminal nucleotideby enzymatically cleaving the terminal internucleoside phosphodiesterbond and reforming same with a pyrophosphate molecule to form a free ATPmolecule according to the following reaction:

NA_(n)+PP_(i)→NA_(n−1)+ATP

catalyzed by poly(A) polymerase. In a quantitative assay for RNA, thedepolymerizing step is repeated essentially to completion or equilibriumto obtain at least two nucleoside triphosphate molecules from a strandof minimally three nucleotides. For detection of DNA, the depolymerizingstep need not be repeated if there are sufficient nucleic acid moleculespresent to generate a signal. The ATP molecules so formed are thendetected with either a luciferase detection system or a NADH detectionsystem. The sensitivity of the reaction may be increased by optionallyamplifying the ATP molecules.

In another embodiment of the present invention, a method is provided forselectively detecting and/or assaying poly(A) mRNA in a reactioncontaining pyrophosphate, adenosine 5′-diphosphate, or a combinationthereof. In this method, a complementary oligo (dT) probe is hybridizedto poly(A) mRNA to form an RNA-DNA hybrid. The oligo (dT) strand of theRNA-DNA hybrid is then depolymerized at the terminal nucleotide byenzymatically cleaving the terminal internucleotide phosphodiester bondand reforming same with a pyrophosphate molecule to form deoxythymidine5′-triphosphate. According to the following reaction:

 TT_(n)+PP_(i)→TT_(n−1)+dTTP

catalyzed by a reverse transcriptase. In a quantitative assay fornucleic acids, the depolymerizing step is repeated essentially tocompletion or equilibrium to obtain at least two nucleotide triphosphatemolecules from a strand of minimally three nucleotides. For detection ofDNA, the depolymerizing step need not be repeated if there aresufficient nucleic acid molecules present to generate a signal. Next,the phosphate groups from the deoxythymidine 5′-triphosphate areenzymatically transferred to adenosine 5′-diphosphate molecules to formATP molecules according to the following reaction:

dTTP*+ADP→dTDP+ATP*

catalyzed by NDPK, wherein P* is the terminal 5′ phosphate sotransferred. Finally, the ATP so formed is detected by a luciferasedetection system or an NADH detection system. If increased sensitivityis desired, the terminal phosphate of the dTTP may be transferred to ADPto form ATP as above followed by an amplification of the resulting ATP.

In another embodiment of the present invention, a method is provided ofdetecting DNA in a reaction containing phosphoribosylpyrophosphate,adenosine 5′-diphosphate, or a combination thereof. In this method, freedeoxyribonucleoside monophosphate molecules are produced from thenucleic acid by digestion with a nuclease. A pyrophosphate group is thenenzymatically transferred from phosphoribosylpyrophosphate molecules tothe deoxyadenosine monophosphate molecules to form deoxyadenosinetriphosphate molecules according to the following reaction:

dAMP+PRPP→dATP+ribose-5-PO₄

catalyzed by phosphoribosylpyrophosphate synthetase. Next, the terminal5′ phosphate groups from the deoxyadenosine triphosphate molecules areenzymatically transferred to adenosine 5′-diphosphate molecules to formATP molecules according to the following reaction:

 dATP*+ADP→dADP+ATP*

catalyzed by NDPK wherein P* is a terminal 5′ phosphate so transferred.The ATP so produced may be detected by a luciferase detection system oran NADH detection system. If desired, the pyrophosphate transferringstep and the phosphate transferring step may be performed in a singlepot reaction. If increased sensitivity is required, the ATP moleculesmay be amplified.

Another embodiment of the present invention provides a method ofdetecting RNA in a reaction containing phosphoribosylpyrophosphate. Freeribonucleoside monophosphate molecules are produced by digestion of RNAwith a nuclease. Next, a pyrophosphate molecule fromphosphoribosylpyrophosphate molecules is enzymatically transferred tothe adenosine monophosphate molecules to form adenosine triphosphatemolecules according to the following reaction:

NMP+PRPP→NTP+ribose-5-PO₄

catalyzed by phosphoribosylpyrophosphate synthetase. The ATP so producedis then detected by a luciferase detection system or an NADH detectionsystem. If increased sensitivity is required, the ATP so produced may beamplified.

Another embodiment of the present invention provides a method fordetermining the presence and/or amount of cells and cellular materialpresent in the sample. In this method, the contents of cells arereleased to form a cell lysate. Phosphate donor molecules and adenosine5′-monophosphate molecules are then added to the cell lysate so thatadenosine 5′-diphosphate molecules are produced by the enzymatictransfer of an phosphate group from the donor to the adenosine5′-monophosphate according to the following reaction:

D-P+AMP→D+ADP

catalyzed by endogenous enzymes present in the cell lysate. ATP is thenproduced by the enzymatic transfer of a phosphate from the donormolecules to adenosine 5′-diphosphate molecules according to thefollowing reaction:

D-P+ADP→D+ATP

also catalyzed by endogenous enzymes present in the cell lysate sample.The adenosine 5′-triphosphate so produced is then detected by either aluciferase detection system or an NADH detection system. The phosphatedonor of this embodiment may be either deoxycytidine 5′-triphosphate,deoxyguanidine 5′-triphosphate, or deoxythymidine 5′-triphosphate.

The present invention further provides a composition of matter forproducing adenosine 5′-triphosphate from DNA, pyrophosphate, andadenosine 5′-diphosphate. This composition comprises a mixture ofnucleoside diphosphate kinase and a nucleic acid polymerase which areprovided in a concentration sufficient to catalyze the production of ATPfrom DNA at about picogram to microgram amounts of DNA.

The present invention, also provides a composition of matter forproducing adenosine triphosphate from DNA, phosphoribosylpyrophosphate,and adenosine 5′-diphosphate. This composition comprises a mixture of aphosphoribosylpyrophosphate synthetase and nucleoside diphosphate kinasein a sufficient concentration to catalyze the production of adenosinetriphosphate from about picogram to microgram amounts of DNA.

The present invention provides various kits for nucleic acid detection.First, a kit is provided which contains reagents for the detection ofDNA by pyrophosphorolysis. The kit contains a vessel containing anucleic acid polymerase and a vessel containing a nucleosidedisphosphate kinase. The nucleic acid polymerase and nucleosidediphosphate kinase may be provided in the same container. Second, a kitis provided which contains reagents for the detection of nucleic acid bynuclease digestion. The kit contains a vessel containingphosphoribosylpyrophosphate synthetase and a vessel containing anuclease. Third, a kit is provided which contains reagents for thedetection of RNA by pyrophosphorolysis. The kit contains a vesselcontaining poly(A)-polymerase. Fourth, a kit containing reagents for thedetection of DNA by nuclease digestion is provided. This kit contains avessel containing phosphoribosylpyrophosphate synthetase and a vesselcontaining nucleoside disphosphate kinase. Thephosphoribosylpyrophosphate synthetase and nucleoside diphosphate kinasemay optionally be provided in the same container.

An embodiment of the present invention further provides a kit containingreagents for the detection of cells and/or cellular material in asample. The kit contains a vessel containing adenosine 5′-monophosphateand a vessel containing a high energy phosphate donor which may not beutilized by luciferase.

The present invention also provides a method of amplifying a nucleosidetriphosphate molecule in a reaction containing adenosine5′-monophosphate molecules, high energy phosphate donor molecules, or acombination thereof. In this method, the terminal 5′ phosphate groupfrom a nucleoside triphosphate molecule (XTP) present in the sample isenzymatically transferred to an adenosine 5′-monophosphate moleculeadded to the sample to form adenosine 5′-diphosphate molecules andnucleoside diphosphate molecules (XTP, either a ribonucleoside ordeoxyribonucleoside triphosphate) according to the following reaction:

XTP+AMP→XDP+ADP

catalyzed by a first enzyme which may be either nucleoside monophosphatekinase or adenylate kinase. Next, a phosphate from a high energyphosphate donor molecule which may not be utilized by the first enzymeis enzymatically transferred to the adenosine 5′-diphosphate moleculesto form adenosine 5′-triphosphate molecules according to the followingreaction:

 ADP+D-P→ATP+D

catalyzed by nucleoside diphosphate kinase or pyruvate kinase. These twosteps are then repeated until the desired level of amplification isachieved. The high energy phosphate donors may be either dCTP or AMP-CPPfor NDPK and PEP for pyruvate kinase.

The present invention also provides a method for detectingdeoxyribonucleic or ribonucleic acid in a reaction containingpyrophosphate, adenosine 5′-monophosphate, and a high energy phosphatedonor, or a combination thereof, in a single pot reaction. First,nucleic acid is depolymerized at a terminal nucleotide by enzymaticallycleaving the terminal internucleotide phosphodiester bond with apyrophosphate molecule to form a free ribonucleoside (XTP) ordeoxynucleoside triphosphate molecule (XTP) according to reaction 1:

NA_(n)+PP_(i)→NA_(n−1)+XTP

catalyzed by a polymerase. The depolymerizing step is repeated to obtainat least two nucleoside triphosphate molecules. The ribonucleosidetriphosphate molecules or deoxyribonucleoside triphosphate molecules arethen amplified by enzymatically transferring the terminal 5′ phosphategroup from the nucleoside triphosphate molecule formed in reaction 1 toan adenosine 5′-monophosphate to produce an adenosine 5′-diphosphatemolecule and a nucleoside 5′-diphosphate molecule (XDP) according toreaction 2 catalyzed by a first enzyme:

XTP+AMP→XDP+ADP.

Next, a phosphate group from a high energy phosphate donor molecule,which is not a substrate for the first enzyme, is enzymaticallytransferred to the adenosine 5′-diphosphate molecules produced in areaction to produce adenosine 5′-triphosphate molecules according toreaction 3 catalyzed by a second enzyme:

ADP+D-P→ATP+D.

The two amplification steps are repeated until the desired level ofamplification is achieved. Enzyme 1 in this method may be eitheradenylate kinase or nucleoside monophosphate kinase, while enzyme 2 maybe either pyruvate kinase or nuceloside diphosphate kinase.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a method for detecting extremely lowlevels of various nucleic acids including both deoxyribonucleic acid(DNA) and ribonucleic acid (RNA) in biological samples, especiallysamples of recombinant proteins. The extreme sensitivity,reproducibility, ease of conducting the reactions, and speed ofconducting the reactions represent major advantages over methodscurrently in use for low level detection of nucleic acids.

The detection method may be divided into three general steps. The firststep is the production of the following nucleosides: nucleosidemonophosphates (XMPs) including the ribonucleoside monophosphates (NMPs)adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP),uridine 5′-monophosphate (UMP), and cytidine 5′-monophosphate (CMP);deoxyribonucleoside monophosphates (dNMPs) including deoxyadenosine5′-monophosphate (dAMP), deoxyguanosine 5′-monophosphate (dGMP),deoxythymidine 5′-monophosphate (dTMP), and deoxycytidine5′-monophosphate (dCMP); nucleoside triphosphates (XTPs) including theribonucleoside triphosphates (NTPs) adenosine 5′-triphosphate (ATP),guanosine 5′-triphosphate (GTP), uridine 5′-triphosphate (UTP), andcytidine 5′-triphosphate (CTP); and the deoxyribonucleosidetriphosphates (dNTPs) including deoxyadenosine 5′-triphosphate (dATP),deoxyguanosine 5′-triphosphate (dGTP), deoxythymidine 5′-triphosphate(dTTP), and cytidine 5′-triphosphate (dCTP). The NMPs and dNMPs areproduced by nuclease digestion and the NTPs and dNTPs bydepolymerization by pyrophosphorolysis. The second step, used when theinitial substrate is DNA, is the transfer of the terminal phosphate fromthe dNTPs to ADP to form ATP. The optional step of XTP amplification maybe performed at this stage to increase the sensitivity of the detectionsystem especially when measuring samples containing low levels of DNA inthe range of 1-10 picograms of nucleic acid. The third step is detectionof ATP by a suitable detection method. Examples of such detectionsystems are the luciferase detection system and NADH-based detectionsystem.

Nucleic acid polymerases generally catalyze the elongation of nucleicacid chains. The reaction is driven by the cleavage of a pyrophosphatereleased as each nucleoside is added. Each nucleoside triphosphate hasthree phosphate groups linked to carbon 5 of ribose or deoxyribose. Theaddition of a nucleoside to a growing nucleic acid results in formationof a internucleoside phosphodiester bond. This bond is characterized inhaving a 3′ linkage to carbon 3 of ribose or deoxyribose and a 5′linkage to carbon 5 of ribose or deoxyribose. Each nucleoside is addedthrough formation of a new 3′ linkage, so the nucleic acid strand growsin a 5′ to 3′ direction. The 5′ end of the nucleic acid is characterizedby a phosphate group attached to carbon 5.

Several polymerases are also known to catalyze the reverse of thepolymerization process. This reverse reaction is calledpyrophosphorolysis. The pyrophosphorolysis activity of DNA polymerasewas demonstrated by Deutscher and Kornberg, Enzymatic Synthesis ofDeoxyribonucleic Acids, J. Biol. Chem. 244: 3019-28 (1969). Othernucleic acid polymerases capable of pyrophosphorolysis include DNApolymerase α, DNA polymerase β, T4 DNA polymerase, Taq polymerase,Klenow fragment, AMV reverse transcriptase, and MMLV reversetranscriptase. However, not all polymerases are known to possesspyrophosphorolysis activity. For example, poly(A) polymerase has beenreported to not catalyze pyrophosphorylation. (See Sippel, Eur. J.Biochem. 37:31-40 (1973).)

A mechanism of pyrophosphorolysis has been suggested for RNA polymerase.It is believed that the partial transfer of a Mg²⁺ ion from theattacking pyrophosphate to the phosphate of the internucleosidephosphodiester bond of the RNA may increase the nucleophilic reactivityof the pyrophosphate and the electrophilicity of the diester asdescribed in Rozovskaya et al. Biochem. J. 224: 645-50 (1984). Theinternucleoside phosphodiester bond is enzymatically cleaved by theaddition of pyrophosphate to the nucleoside 5′ phosphate and a newphosphodiester bond is formed between the pyrophosphate and thenucleoside monophosphate.

The pyrophosphorolysis reaction can be summarized as follows:

NA_(n)+PP_(i)→NA_(n−1)+XTP  Reaction 1

wherein NA is a nucleic acid, PP_(i) is pyrophosphate and XTP is eithera deoxyribonucleoside triphosphate molecule or a ribonucleosidetriphosphate molecule. The reaction may then be repeated to obtain atleast two XTP molecules. It should be noted that the reaction may berepeated on the same nucleic acid molecule or on a plurality ofdifferent nucleic acid molecules.

Preferred reaction mixes for depolymerization by pyrophosphorolysis,including suitable buffers for each nucleic acid polymerase analyzed aredisclosed in the Examples. Under these conditions, sufficient NTP ordNTP is produced to accurately detect or assay extremely low amounts ofnucleic acids (5-15 picograms.)

Even though the preferred reaction conditions for polymerization anddepolymerization by pyrophosphorolysis are similar, the rates of thesereactions vary greatly. For example, AMV and RLV reverse transcriptasescatalyze pyrophosphorolysis under optimal conditions at a rate of aboutfifty- to one hundred-fold less than polymerization as demonstrated inSrivastavan and Modak, J. Biol. Chem. 255 (5): 2000-04 (1980). Thus, thehigh efficiency of the pyrophosphorolysis reaction was unexpected andappears to be associated with extremely low levels of DNA substrate incontrast to previous DNA pyrophosphorolysis studies conducted on muchhigher amounts of DNA.

The pyrophosphorolysis activity of different nucleic acid polymerasesalso varies. T4 polymerase appears to possess the greatestpyrophosphorolysis activity as measured by a luciferase assay for ATPproduced by pyrophosphorolysis. Pyrophosphorolysis using T4 polymeraseresulted in about a 10 fold increase in light production as compared toMMLV-RT and a 4 fold increase in light production as compared to Taqpolymerase.

The detection of nucleic acids at low picogram levels is generallyenhanced by fragmenting or partially digesting the nucleic acid.Preferably, fragmentation is accomplished by sonication or restrictionenzyme digestion of the nucleic acid, forming a plurality of smallernucleic acid fragments. This step probably enhances detection becausethe pyrophosphorolysis reaction only proceeds from the DNA ends, asdemonstrated in the Examples. Providing a greater number of DNA endsmeans that more reactions are occurring at any one time. It should benoted that DNA ends may be present within a molecule as well as at theend of a linear DNA fragment. For example, polymerases may catalyzepyrophosphorolysis from a gap in a DNA segment or a nick in a DNAsegment. The type of enzyme used for pyrophosphorolysis and the type ofsubstrate determine whether fragmentation is necessary. For instance,the data set forth in the Examples demonstrate that fragmenting greatlyincreases detection of plasmid DNA when Taq polymerase is used, but doesnot effect detection when T4 polymerase is used. However, whenchromosomal DNA is the substrate, fragmentation increases detection fromboth enzymes.

The type of cut made by restriction enzyme digestion also affects thepyrophosphorolysis activity of different nucleic acid polymerases. Forexample, MMLV-RT and Taq polymerase can catalyze pyrophosphorolysis ofDNA fragments with 5′ overhangs, but not 3′ overhangs. In contrast, T4DNA polymerase catalyzes both 3′ and 5′ end overhang and blunt endmediated pyrophosphorolysis. T4 polymerase is thus a preferred enzymefor pyrophosphorolysis. When other nucleic acid polymerases are utilizedfor pyrophosphorolysis of restriction enzyme treated DNA, care must betaken to match the overhang specificity of the polymerase with the typeof overhang created by the restriction endonuclease.

It must be noted that sequence specificity of pyrophosphorolysis forsingle stranded DNA has been previously noted during sequencing by Taborand Richardson, J. Biol. Chem. 265 (14): 8322-28 (1990). The sequencespecificity of the pyrophosphorolysis reaction was noted when somedideoxynucleoside terminated sequence fragments were shown to be moresusceptible to degradation by phosphorolysis than other fragments.

Further, the type of polymerase used in the pyrophosphorolysis reactionmust be matched to the correct nucleic acid substrate. In general, DNApolymerases and reverse transcriptases are preferred for depolymerizingDNA, and RNA polymerases are preferred for depolymerizing RNA. Reversetranscriptases are preferred for depolymerizing RNA-DNA hybrids.

Applicants have further demonstrated that poly(A) polymerase maycatalyze pyrophosphorolysis, even though no such reaction has beenpreviously reported. In fact, poly(A) polymerase has been widelyreported to not catalyze pyrophosphorolysis. See, for example, Sippel,Eur. J. Biochem. 37:31-40 (1973) and Sano and Feix, Eur. J. Biochem.71:577-83 (1976). Surprisingly, the applicants show that under theproper reaction conditions poly(A) polymerase catalyzes phosphorolysis.Preferably, the manganese chloride present in the previously reportedbuffers is omitted, the concentration of sodium chloride is decreased,and the pH is lowered from about 8.0 to about 7.5. Most preferably,poly(A) polymerase pyrophosphorolysis reaction buffer contains about 50mM Tris-Cl pH 7.5, 10 mM MgCl₂, 50 mM NaCl, and 2 mM NaPP_(i) (sodiumpyrophosphate).

It is important to note that the depolymerization reaction is thereverse of the polymerization reaction. Therefore, as increasing amountsof free nucleoside triphosphates are produced by depolymerization, astate of equilibrium may theoretically be attained in whichpolymerization and depolymerization reactions are balanced.Alternatively, where small amounts of nucleic acid are detected, thereaction may go essentially to completion without reaching equilibrium,i.e. the nucleic acid depolymerized into its constituent subunitsnucleotides by greater than 90%. This is important in a quantitativetest because the total amount of nucleotides released is proprtional tothe amount of signal generated in the detection assay. When used forqualitative detection of nucleic acid, it is not necessary that thereaction reach equilibrium or go essentially to completion provided athreshold level of nucleotides are produced. The mixture of nucleosidetriphosphate molecules produced by depolymerization will preferably beconverted to adenosine triphosphate as described below. For eitherdetection or assay, a detectable threshold level of 6×10⁷ adenosinetriphosphate molecules must be provided for detection of light from atypical luciferase assay.

In a preferred embodiment of the present invention for detecting nucleicacids, nucleic acid polymerase and PP_(i) are added to a samplecontaining less than 1 μg nucleic acid, down to less than about 10 pg ofnucleic acid. To increase the sensitivity of the DNA detection, the DNAmay be fragmented by treatment with a restriction endonuclease or bysonication. Next, the nucleic acid is degraded by pyrophosphorolysisreleasing free NTPs or dNTPs. Enzymes useful in the pyrophosphorolysisreaction include AMV reverse transcriptase, MMLV reverse transcriptase,DNA polymerase alpha and beta, Taq polymerase, T4 DNA polymerase, Klenowfragment and poly(A) polymerase. Most preferably, T4 polymerase isutilized for DNA pyrophosphorolysis reactions because of its recognitionof 3′ and 5′ overhangs and blunt ends and high processivity as notedabove.

Luciferase, which is part of the preferred ATP detection system, isinhibited by pyrophosphate (PP_(i)). Therefore, care must be taken notto transfer a highly inhibiting amount of PP_(i) to the ATP detectionreaction. Preferably, the amount PP_(i) carried over to the ATPdetection reaction results in a concentration of PP_(i) in theluciferase detection reaction of less than about 100 μM, although lessthan about 10 μM is desirable. Therefore, the amount of PP_(i) utilizedin the pyrophosphorolysis reaction will be determined by the size of thealiquot which is taken for use in the luciferase detection system. Thealiquot size may vary, but the amount of PP_(i) transferred or carriedover to the luciferase detection reaction should correspond to thePP_(i) concentration parameters described above so that theconcentration of PP_(i) is at least below about 100 μM, and preferablybelow about 10 μM.

In another embodiment, the nucleic acids may be first degraded into NMPor dNMP by exonuclease digestion according to the following reaction:

NA_(n)+H₂O→NA_(n−1)+XMP  Reaction 2

wherein NA is a nucleic acid, XMP is either a deoxyribonucleosidemonophosphate or ribonucleoside monophosphate, and n is the number ofnucleosides in the nucleic acid.

Nuclease digestion may be accomplished by a variety of nucleasesincluding S1 nuclease, nuclease BAL 31, mung bean nuclease, exonucleaseIII and ribonuclease H. Nuclease digestion conditions and buffers may befound in the Product Literature available from commercial sources, or asdisclosed in the Examples.

After digestion with the nuclease, the NMPs or dNMPs are converted toNTPs or dNTPs respectively. U.S. Pat. No. 4,375,897 describes thedetection of RNA by digestion with nucleases followed by conversion toNTP. This method utilizes a two-step scheme in which adenylate kinaseconverts AMP to ADP, and pyruvate kinase then converts ADP to ATP. Thismethod is essentially limited to the detection of poly(A) mRNA becauseno mechanism is suggested for conversion of dNTPs to ATP, the preferredsubstrate for luciferase. Nuclease digestion or phosphorolysis of DNAresults in a mixture of dNTPs which do not act as efficient substratesfor luciferase.

In the biosynthesis of purine and pyrimidine mononucleosides,phosphoribosylpyrophosphate (PRPP) is the obligatory ribose-5′-phosphatedonor. PRPP itself is formed in a reaction catalyzed by PRPP synthetasethrough the transfer of pyrophosphate from ATP to ribose-5-phosphate.This reaction is known to be reversible as described in Sabina et al.,Science 223: 1193-95 (1984).

In the present invention, the NMP or dNMP produced by nuclease digestionis preferably converted directly to NTP or dNTP by the enzyme PRPPsynthetase in the following reactions:

XMP+PRPP→XTP+ribose-5-PO₄  Reaction 3

wherein XMP is either adenosine monophosphate or deoxyadenosinemonophosphate and XTP is either a adenosine triphosphate ordeoxyadenosine triphosphate. Preferably, this reaction produces adetectable threshold level of at least 6×10⁷ adenosine triphosphatemolecules.

In this reaction, the pyrophosphate group of PRPP is enzymaticallytransferred to XMP molecules, forming XTP molecules. Reaction conditionsand buffers are set forth in the Examples. When RNA is the substrate,the ATP produced may be directly detected.

Utilization of the PRPP reaction in the nucleic acid detection systemhas several advantages over the prior art. First, only one step isnecessary to convert an AMP or dAMP to a ATP or dATP, which simplifiesthe detection system. Second, contamination of the detection reactionwith exogenous ATP, ADP, or AMP is less likely.

The dNTPs produced by pyrophosphorolysis or nuclease digestion followedby pyrophosphorylation can theoretically be used directly as substratesfor luciferase, allowing detection of the nucleic acid. However, thepreferred substrate for luciferase is ATP as demonstrated by Moyer andHenderson, Nucleoside Triphosphate Specificity of Firefly Luciferase,Anal. Biochem. 131:187-89 (1983). When DNA is the initial substrate, anucleoside diphosphate kinase (NDPK) is conveniently utilized tocatalyze the conversion of dNTPs to ATP by the following generalreaction:

dNTP*+ADP→dNDP+ATP*  Reaction 4

wherein dNTP is a mixture of deoxyribonucleoside triphosphates and dNTPis the corresponding deoxyribonucleoside triphosphate. In the reaction,the terminal 5′-triphosphate (P*) of the dNTP is transferred to ADP toform ATP.

Enzymes catalyzing this reaction are generally known as nucleosidediphosphate kinases (NDPK). NDPKs are ubiquitous, relatively nonspecificenzymes. For a review of NDPK, see Parks and Agarwal, in The Enzymes,Volume 8, P. Boyer Ed. (1973). The conversion of NTPs or dNTPs to ATP byNDPK is preferably accomplished by adding NDPK and a molar excess of ADPover the amounts of NTPs or dNTPs expected to be produced bypyrophosphorolysis, or nuclease digestion followed bypyrophosphorylation by PRPP synthetase. Alternatively, if anamplification scheme is used, a molar excess of AMP may be used as thepreferred substrate. The utilization of ADP requires optimization of theamount of ADP added. Too much ADP results in high background levels. Areaction containing NDPK contains about 0.01 to 0.50 μM ADP, preferablyabout 0.05 μM ADP. Illustrative buffers and reaction components are setforth in the Examples.

As an optional step, the NTP, dNTP, or ATP generated by thepyrophosphorolysis or nuclease digestion schemes may be amplified togive even greater sensitivity. Amplification may be required whenutilizing detection systems other than luciferase or when increasedlevels of signal are needed for detection by a less sensitiveluminometer. Amplification of NTP means a continuous reaction wherein 1NTP gives rise to 2 NTPs, which can be cycled to yield 4 NTPs and so on.When AMP is added to feed the amplification reaction, ATP willaccumulate while the amount of original NTP remains the same. PCTpublication WO 94/25619 and Chittock et al., Anal. Biochem., 255:120-6(1998), incorporated herein by reference, disclose amplification systemsfor ATP characterized by the following coupled reactions:

C1+S1→2C2 and 2C2+2S2→2C1

2C1+2S1→4C2 and 4C2+4S2+E2→4C1

4C1+4S1→8C2 and 8C2+8S2+E2→8C1  Reaction 5

wherein C1 is the target compound present in a sample to be amplified,S1 is the amplification substrate, E1 is a catalytic enzyme capable ofutilizing C1 and S1 to produce C2, S2 is an energy donating substrate,and E2 is a catalytic enzyme capable of utilizing C2 and S2 to produceC1, which then recycles through the reaction. According to this reactionscheme, each pass through the coupled reaction doubles the amount of C1,which can be subsequently detected. Patent Application GB 2,055,200discloses an amplification system utilizing adenylate kinase andpyruvate kinase.

In designing a coupled ATP amplification reaction for use in nucleicacid detection, two main requirements must be considered. First, E1 mustnot be able to utilize the high energy phosphate donor utilized by E2.If E1 can utilize the high energy phosphate donor, the ATP amplificationreaction would proceed in the absence of NTP or dNTP produced as aresult of pyrophosphorolysis or nuclease digestion followed bypyrophosphorylation. This would result in the undesirable occurrence offalse positive results. Second, a molar excess of the added high energyphosphate donor must be provided as compared to the amount of dXTP orXTP expected in the reaction. Third, E1 must be able to utilize eitherthe NTP, dNTP, or ATP produced in step 1 by pyrophosphorolysis ornuclease digestion of the nucleic acid.

The amplification system of the present invention may be characterized,as follows:

 XTP+AMP→XDP+ADP

ADP+D-P→ATP  Reaction 6

wherein D-P is a high energy phosphate donor and E1 and E2 are enzymescapable of catalyzing the transfer of phosphates from an XTP to AMP andfrom the D-P to ADP, respectively. The ATP so produced may reenter thereaction (as XTP) and the reaction repeated until the substrates areexhausted or equilibrium is reached, resulting in the production of twoATPs for every ATP supplied to or generated by the reaction. Note thatwhen the target XTP is any nucleoside triphosphate other than ATP theinitial pass through the cycle yields only 1 ATP which then reenters thecycle to produce two ATP, which reenter the cycle to produce 4 ATP andso on. Preferably, the amplification reaction produces a detectablethreshold level of 6×10⁷ adenosine triphosphate molecules.

The XTP in reaction 6 is a ribonucleoside triphosphate ordeoxyribonucleoside triphosphate, which may preferably be ATP providedby pyrophosphorolysis (Reaction 1) or created from XTP by NDPKconversion of ADP to ATP (Reaction 5) or provided by nuclease digestioncoupled with pyrophosphorylation (Reaction 4) followed by NDPKconversion to ATP (Reaction 5). It must be appreciated, however, thatwhen an amplification step is utilized for a DNA substrate, the step ofconverting dNTP to ATP is inherent in the amplification system.Therefore, a separate converting step is not needed.

A nucleoside monophosphate kinase (NMPK) or adenylate kinase ispreferably utilized as enzyme 1 (E1). NMPKs occur as a family, each ofwhich is responsible for catalyzing the phosphorylation of a particularNMP. Until recently, it was generally thought that ATP and dATP werepreferred phosphate donors. However, Shimofuruya and Suzuki Biochem.Intl. 26 (5):853-61 (1992) recently demonstrated that at least someNMPKs can utilize other phosphate donors such as CTP and UTP. Enzyme 2(E2) is preferably nucleoside diphosphate kinase (NDPK) or pyruvatekinase. NDPK's generally catalyze the transfer of the terminal5′-triphosphate of NTPs to NDPs to form NTPs. Pyruvate kinase catalyzesthe transfer of phosphate from phosphoenolpyruvate to ADP to form ATP.These enzymatic activities are utilized in the amplification reaction totransfer a phosphate group from a high energy phosphate donor (D-P) toeither ADP or an NDP.

A high energy phosphate donor (D-P) that may be used by E2 but not by E1is required. When E2 is NDPK, dCTP or α,β methylene adenosine5′-triphosphate (AMP-CPP) may be utilized as D-P. When E2 is pyruvatekinase, phosphoenol pyruvate (PEP) is the preferred high energyphosphate donor. The ability of NDPK to utilize these substrates atefficiencies allowing production of minute quantities of ATP was notknown. It is surprising that these high-energy phosphate donors utilizedwith NMPK and adenylate kinase meet the requirements of theamplification reaction when the recent literature suggests that NMPK(E1) may utilize phosphate donors other than ATP or dATP. Thenonspecificity of adenylate kinase is also well known. The high energyphosphate donor and/or AMP must be provided in a molar excess ascompared to the amount of ATP or dNTP expected present in the sample sothat the high energy phosphate donor is not recycled at an appreciablerate. Additional buffers and reaction components are included in theExamples.

The third step of nucleic acid detection is detection of the NTP, dNTPor amplified ATP. Two well known detection systems include: the lightemitting luciferase detection system, and the NADH light adsorptiondetection system (NADH detection system).

The ATP so produced is detected by a luciferase detection system. In thepresence of ATP and O2, luciferase catalyzes the oxidation of luciferin,producing light which can then be quantitated using a luminometer.Additional products of the reaction are AMP, pyrophosphate andoxyluciferin. The light can be detected by a luminometer.

The preferred ATP detection buffer, which will be referred to as LARbuffer, is formulated by mixing 19.1 ml of deionized water; 800 μl of0.5M Tricine, pH 8.0; 70 μl of 1M MgSO₄; 4 μl of 0.5M EDTA; 0.108 g ofDTT (dithiothreitol); 0.003 g of Luciferin; and adjusting the pH to 7.8if necessary. Preferably, about 5 to 10 nanograms of recombinantluciferase (Promega Lot 6414002) is used in the reaction. Greateramounts of luciferase have a tendency to increase non-specificbackground. Applicants also have shown that deleting coenzyme A from theLAR reaction mix decreases background.

In the NADH detection system, a combination of two enzymes,phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase, areused to catalyze the formation of NAD from NADH in the presence of ATP.ATP is measured as a loss in fluorescence intensity because NADH isfluorescent while NAD is not. Examples of NADH based ATP assays aredisclosed in U.S. Pat. Nos. 4,735,897, 4,595,655, 4,446,231 and4,743,561, and UK Patent Application GB 2,055,200, all incorporatedherein by reference.

Certain of the above reactions may be performed as single pot reactions.A single pot reaction is a reaction wherein at least two enzymes (E1 andE2) with catalytic activity are present in the same reaction mix and acton one or more substrate(s) (S1 and S2). The reactions catalyzed by theenzymes may occur simultaneously where E1 acts on S1 and E2 acts on S2successfully. Alternatively, the reactions catalyzed by E1 and E2 mayoccur in a step-wise or coupled manner where E1 acts on S1 to produce anintermediate S2_(i) and E2 then acts on S2_(i). Of course, such acoupled reaction may also be essentially simultaneous.

The ability to utilize combinations or mixtures of the enzymes of thepresent invention in single pot reactions is surprising in light of theextremely low levels of nucleic acid detection which are achieved. Thislow level detection is possible even though some enzymes are used underless than optimal conditions. As previously described, it was necessaryto optimize the concentration of PP_(i) utilized in thepyrophosphorolysis reactions so that luciferase would not be inhibited.Therefore, aliquots from the NMP, dNMP, NTP, dNTP and ATP producingreactions may be directly added to LAR buffer for luciferase detectionwithout any purification of the reaction products. The luciferasereaction is not poisoned or otherwise quenched by the components of thereactions. This desirable feature allows high throughput screening witha minimal amount of time and effort, and also allows great flexibilityin the design of the overall detection schemes.

Preferably, the pyrophosphorolysis reaction producing dNTP and the NDPKcatalyzed reaction in which the NTPs or dNTPs are converted to ATP maybe performed in a single pot reaction in the nucleic acid polymerasebuffer. NDPK activity is sufficient to convert dNTP to ATP even thoughthe polymerase buffer conditions are suboptimal for NDPK activity. Thepolymerase enzyme and NDPK may both be present initially in thereaction, or the NDPK may be added directly to the reaction after anincubation period sufficient for the production of NTP or dNTP. Anucleic acid polymerase and NDPK may be provided in the same vessel ormixture for use in the reactions described above. The mixture preferablycontains the nucleic acid polymerase and NDPK in a concentrationsufficient to catalyze the production of ATP when in the presence of anucleic acid, pyrophosphate and ADP. Preferably, the polymerase isprovided in a concentration of about 1 to 100 units/μl, most preferablyat about 5 units/μl. Preferably, the NDPK is provided in a concentrationof 0.1 to 100 units/μl, most preferably at about 5 units/μl. Preferably,the mixture is greater than 99% pure.

Similarly, the PRPP synthetase and NDPK reactions can be performed in asingle pot reaction in the PRPP synthetase buffer. Again, NDPK activityis sufficient even though conditions for NDPK activity are suboptimal.The nuclease digested sample containing free NMPs and dNMPs may be addedto a reaction mix initially containing PRPP synthetase and NDPK, oradded to a PRPP synthetase reaction followed by addition to a reactionmix containing NDPK and the luciferase detection reaction components.The preferred buffers and reaction components may be found in theExamples. PRPP synthetase and NDPK may be provided in the same vessel ormixture for use in the reactions described above. The mixture preferablycontains the PRPP synthetase and NDPK in a concentration sufficient tocatalyze the production of ATP when in the presence ofphosphoribosylpyrophosphate and ADP. Preferably, the NDPK is provided ina concentration of 0.1 to 100 units/μl, most preferably at about 5units/μl. Preferably, the PRPP synthetase is provided in a concentrationof 0.001 to 10 units/μl, most preferably at about 0.01 units/μl Ifamplification is desired, the PRPP synthetase reaction must be heatinactivated, otherwise the PRPP synthetase would convert the added AMPto ATP. Preferably the mixture is greater than 99% pure.

The pyrophosphorolysis reaction and amplification reaction may also beperformed in a single pot reaction. In this single pot reaction, thepolymerases may be AMV reverse transcriptase, MMLV reversetranscriptase, DNA polymerase alpha or beta, Taq polymerase, T4 DNApolymerase, Klenow fragment or poly(a) polymerase, a first enzyme forconverting AMP to ADP may be myokinase (adenylate kinase) or NMPK, and asecond enzyme for converting ADP to ATP may be pyruvate kinase or NDPK.The reaction must be fed AMP, preferably Apyrase treated AMP so thatbackground due to contaminating ADP and ATP is minimized. Preferably 1μl of 1U/μl Apyrase may be added to 19 μl of 10 mM AMP, followed byincubation at room temperature for 30 minutes and heat inactivation ofthe Apyrase by incubation at 70° C. for 10 minutes. High energyphosphate donors must also be added to the reaction. When pyruvatekinase is utilized phosphoenolpyruvate is added, while when NDPK isutilized dCTP is added. Preferably, the high energy phosphate donor isadded about 15 minutes after a preincubation with the polymerase,although this is not necessary. These reactions may characterized asfollows:

NA_(n)+PP_(i)→NA_(n−1)+XTP

XTP+AMP→ADP+XDP

ADP+D-P→ATP+D  Reaction 7

wherein NA is a nucleic acid, XTP is a nucleoside triphosphate, either adeoxynucleoside or ribonucleoside triphosphate, XDP is a nucleosidediphosphate, either a deoxynucleoside or ribonucleoside diphosphate, andD-P is a high energy phosphate donor. It should be appreciated that thisreaction produces ATP, the preferred substrate for luciferase, fromdNTPs. The amplification reaction proceeds as described in reaction 7 toproduce a detectable threshold level of 6×10⁷ adenosine triphosphatemolecules. Preferably, the polymerase is provided in a concentration ofabout 1 to 100 units/μl, most preferably at about 5 units/μl.Preferably, the NDPK is provided in a concentration of 0.1 to 100units/μl, most preferably at about 1 unit/μl. Preferably, thePreferably, the mixture is greater than 99% pure.

In another embodiment, the reactions described above may be used toselectively detect poly(A) mRNA according to the following scheme. Firstoligo(dT) primers are hybridized to the poly(A) tails of the mRNA toform a DNA-RNA hybrid. Next, a pyrophosphorolysis reaction is performedusing reverse transcriptase (RT). Reverse transcriptases which may beused in the present invention include Mouse Mammary Leukemia Virus(MMLV) RT, Avian Myeloma Virus (AMV) RT and Rous Sarcoma Virus (RSV) RT.An advantage of this detection system is that these RTs catalyzepyrophosphorolysis of double stranded nucleic acid and double strandedRNA-DNA hybrids, but not single stranded nucleic acids. Thus, the amountof poly(A) RNA in a total cellular RNA sample be determined. Thepyrophosphorolysis reaction produces dTTP according to the followingreaction:

TT_(n)+PP_(i)→TT_(n−1)+dTTP;  Reaction 8

wherein TT_(n) is oligo(dT) and PP_(i) is pyrophosphate.

The dTTP can be converted to ATP by NDPK as described in reaction 4above, optionally amplified, and detected as described above.

In another embodiment, the reactions described above may be used todetect the presence of cells in a sample. U.S. Pat. No. 5,648,232,incorporated herein by reference, describes a method of detecting cellsin a sample. That method takes advantage of adenylate kinase activity,which is present in all living organisms. Briefly, a sample suspected ofcontaining microorganisms or other living cells is subjected toconditions causing cell lysis. ADP is then added to the lysate, which isconverted by endogenous adenylate kinase activity to ATP by thefollowing reaction:

ADP+ADP→ATP+AMP  Reaction 9

The ATP produced by this reaction is then detected by the luciferaseassay system.

The present invention also provides a method of detecting the presenceof cells in a lysate of a sample suspected of containing cellularmaterial by using different substrates. This system takes advantage of acoupled reaction catalyzed by endogenous adenylate kinase activity (AK)and NDPK activity according to the following reaction scheme:

AMP+D-P→D+ADP and

ADP+D-P→ATP+D.  Reaction 10

wherein D-P is a high energy phosphate donor added to the cell lysateand AMP is adenosine monophosphate added to the cell lysate sample. Inthis reaction, adenosine 5′-diphosphate molecules are produced by theenzymatic transfer of a phosphate group from the high energy phosphatedonor molecules (D-P) to the added adenosine 5′-monophosphate (AMP)molecules. Then, adenosine 5′-triphosphate is produced by the enzymatictransfer of phosphate from D-P molecules to the adenosine 5′-diphosphatemolecules according to the following general reaction catalyzed byendogenous enzymes present in the cell lysate sample

Co-optimization of the concentrations of nucleosides added to thesamples was necessary to optimize light output from these reactions.About 1 mM to 80 mM AMP and 1 mM to 100 mM dCTP may be added to the testsample, and preferably about 10 mM AMP and 100 mM dCTP may be added tothe test sample. After addition of nucleosides to the sample, thesamples are preferably incubated at room temperature for about 10 to 60minutes, and light output from the samples determined by a luminometer.Other preferred buffers and reactions components may be found in theExamples.

This system has an important advantage over previously described celldetections systems. The AMP and dCTP are much more stable than ADP, sothe results are more reproducible.

In another aspect of the present invention, a nucleic acid detectiontest kit is provided for performing the pyrophosphorolysis nucleic aciddetection method. The nucleic acid detection test kit comprises theessential reagents required for the method of the nucleic acid detectioninvention. For nucleic acid detection by pyrophosphorolysis, the kitincludes a vessel containing an enzyme capable of catalyzingpyrophosphorolysis such as Taq polymerase, T4 polymerase, AMV reversetranscriptase, MMLV reverse transcriptase, or poly(A) polymerase. Theconcentration of polymerase is 0.1 to 100 units/μl, preferably about 5units/μl. Kits for use in DNA detection also include a vessel containingnucleoside diphosphokinase and a vessel containing ADP. Preferably,these reagents are free of contaminating ATP and adenylate kinase. TheNDPK is provided in concentration of about 0.1 to 100 units/μl,preferably about 1.0 units/μl. The contaminants may be removed bydialysis or Apyrase treatment. Optionally, the kit may contain vesselswith reagents for amplification of dNTPs or NTP to ATP. Amplificationreagents include pyruvate kinase, adenylate kinase, NMPK, NDPK, AMP asthe amplification substrate, and dCTP or AMP-CPP as high-energyphosphate donors. The kit may be packaged in a single enclosureincluding instructions for performing the assay methods. The reagentsare provided in containers and are of a strength suitable for direct useor use after dilution. A standard set may also be provided to allowquantitation of results. Test buffers for optimal enzyme activity may beincluded. Most preferably, the NDPK and nucleic acid polymerase areprovided in the same reaction mix so that a single pot reaction may beperformed consistently.

In another aspect of the present invention, a nucleic acid detection kitis provided for performing the nuclease digestion nucleic acid detectionmethod of the present invention. This test kit comprises the essentialreagents required for this method. These reagents include a nuclease,PRPP synthetase, PRPP, NDPK, and ADP together with luciferase andluciferin. The nuclease is provided in a concentration of about 1 to 500units/μl, preferably about 20 units/μl. The PRPP synthetase is providedin concentration of about 0.01 units/μl to 10 units/μl, preferably about0.1 units/μl. Preferably, the kit includes all these reagents withluciferase and luciferin being provided as a single reagent solution.Most preferably, the PRPP synthetase and NDPK are provided in a singlereaction mix so that a single pot reaction containing these two enzymesmay be performed, simplifying the detection method. The kit is in theform of a single package preferably including instructions to performthe method of the invention. The reagents are provided in vessels andare of a strength suitable for direct use or use after dilution.Preferably, buffers which support the optimal enzyme activity areprovided. Optionally, reagents for amplification of the ATP signal maybe provided as described in the previous kit.

In another aspect of the present invention, a test kit is provided fordetermining the presence of microorganisms or other cells in a testsample. This test kit comprises the essential reagents required for themethod. These reagents include a highenergy phosphate donor which maynot be utilized by luciferase, preferably dCTP, and AMP together withluciferase and luciferin. Preferably, the kit includes all thesereagents with luciferase and luciferin being provided in the samesolution. Preferably, the reagents are free of contaminating componentssuch as adenylate kinase and ATP that would cause a false positive test.A cell lysis cocktail may be provided for efficiently releasing thecontents of the target cells for each of the assays intended. Forprokaryotic microorganisms, only a cationic detergent is needed. Forfungal spores or eukaryotic cells assays, a further nonionic detergentreagent is included. Reagents are provided in vessels and are of astrength suitable for direct use or use after dilution. A buffersolution for diluting the cell samples may also be provided.

Other aspects of the present invention will be made apparent in thefollowing examples. These Examples are intended to illustrate theinvention and in no way limit any aspect of the invention.

EXAMPLES Example 1 Detection of ATP Using Luciferase

The ultimate sensitivity of detection using an enzyme based detectionsystem is related to the ability of the enzymatic reaction to produce ameasurable signal over background. This example describes the detectionof very low levels of ATP using Luciferase.

Luciferase Assay Reagent buffer (LAR) was produced by mixing: 19.1 ml ofnanopure water; 800 μl of 0.5M Tricine (Sigma T9784), pH 8.0; 70 μl of1M MgSO₄ (Promega AA319, Lot #970931); 4 μl of 0.5M EDTA (Promega AA189,Lot #962131); 0.13 g of DTT (dithiothreitol, Promega V31SA); 0.003 g ofBeetle Luciferin (Promega E160C, Lot #79838); 0.0044 g of Coenzyme A, pH7.8 (Pharmacia 28-3001-03 Lot #7053001031). A 0.1M solution of ATP wasprepared by dissolving solid ATP (Sigma A9187) in Tris 10 mM pH 7.5.This stock solution was diluted into Tris 10 mM pH 7.5 to producesolutions at 100 μM, 1 μM, 10 nM, and 100 pM. Recombinant Luciferase(Promega #1701, Lot #6414002) was diluted to 1 mg/ml, 100 μg/ml, and 10μg/ml using nanopure water.

Reactions were assembled in duplicate containing the followingcomponents in 1.5 ml polypropylene tubes as described in Table 1.

TABLE 1 Components Reaction LAR Luciferase Total Luciferase Added 1 50ul 1 ul of 1 mg/ml 1 μg 2 50 ul 1 ul of 100 μg/ml 100 ng  3 50 ul 1 ulof 10 μg/ml 10 ng  4 50 ul (none) 0 ng

Immediately upon addition of the Luciferase, the tube was read in aTurner TD-20e Luminometer. The values obtained are listed in Table 2.

TABLE 2 Reaction Light Units Light 2 Avg. Filter* Total Light Units 190.6 66.09 78.345 286 22406.67 2 2048 2096 2072 none 2072 3  148  122 135 none  135 4   0   0   0 none   0 *= light reduction filter used toreduce signal, light units measured must be multiplied by filter toobtain light output

Luciferase requires both ATP and Luciferin to produce a light signal.The light produced in the reactions above is the result of ATPcontamination of either the LAR reagent or the Luciferase added to thereactions. Ten and five nanogram levels of Luciferase were chosen forfurther studies since they produced the lowest level of background lightwithout ATP addition, yet were expected to give greatly increased lightoutput upon addition of ATP.

Reactions were assembled in duplicate containing 50 μl of LAR buffer, 1μl of stock Luciferase providing either 5 or 10 ng of luciferase to thereaction, and ATP at the concentrations listed below. Light output fromreactions was then immediately determined with a Turner TD-20eLuminometer. The results are described in Table 3. These data indicatethat Luciferase is capable of detecting low levels of ATP if levels ofLuciferase are used that minimize the background resulting from ATPcontamination of the reagent.

TABLE 3 ATP Luciferase (ng) Conc. μl Light Units Filter Avg. Light Units10 (none) na 131.7 119.6 (none) 125.7 10 100 uM 5 26.09 25.25 286 7221.510  1 uM 5 249.5 226.1 (none) 237.8 10  10 nM 5 131.7 232.6 (none) 182.210 100 pM 5 215.4 143.9 (none) 179.7  5 (none) na 52.76 50.04 (none)51.4  5 100 uM 5 17.99 18.47 286 5213.8  5  1 uM 5 156.4 174.4 (none)167.9  5  10 nM 5 46.13 34.37 (none) 40.3

Example 2 Limit of ATP Detection Using Luciferase

This example demonstrates that the light output values obtained fromreactions with very low levels of ATP are statistically different fromappropriate control reactions. The limit of detection can be defined asthe amount of the analyte that generates a signal which has less than a0.05 probability of identity to the data from control reactions usingthe Student's t-Test.

ATP (Sigma A9187, Lot #36H7808 Promega, stored overnight at −20 degrees)in 10 mM Tris-Cl pH 7.5 was diluted to 500 nM and 50 nM. Various amountsof ATP were added to 350 μl LAR, with Tris added to make up thedifference in volume (385 total). Only Tris was added to the control todetermine the background signal. After mixing, 6 aliquots of 50 μl ofthe control and containing samples were transferred to luminometertubes. Luciferase (2 μl of 2.5 ng/μl in 1×CCLR with 1 mg/ml BSA (1×CCLRPromega E153A, Lot #7903201)) was added to the reaction, the tube wastapped to mix the reagents and light output was immediately determinedwith the Turner TD-20e luminometer. The data is presented in Table 4.

TABLE 4 Reaction ATP (M) Light  1 0 2.744  2 0 2.606  3 0 2.849  4 02.834  5 0 2.801  6 0 2.778  7 4.5 × 10⁻¹⁰ 4.883  8 4.5 × 10⁻¹⁰ 5.192  94.5 × 10⁻¹⁰ 4.945 10 4.5 × 10⁻¹⁰ 4.220 11 4.5 × 10⁻¹⁰ 5.282 12 4.5 ×10⁻¹⁰ 5.216 13 9.1 × 10⁻¹⁰ 7.167 14 9.1 × 10⁻¹⁰ 8.100 15 9.1 × 10⁻¹⁰7.774 16 9.1 × 10⁻¹⁰ 8.047 17 9.1 × 10⁻¹⁰ 8.010 18 9.1 × 10⁻¹⁰ 7.677 191.82 × 10⁻⁹ 10.70 20 1.82 × 10⁻⁹ 11.02 21 1.82 × 10⁻⁹ 11.93 22 1.82 ×10⁻⁹ 11.91 23 1.82 × 10⁻⁹ 12.27 24 1.82 × 10⁻⁹ 11.92

The Student's t-Test (a 2-tailed test for 2 samples with unequalvariance) was used to analyze the data. The light output from each ATPconcentration was compared to the light output of the backgroundcontrol, and a p-value determined for each comparison. The results ofthe analysis are presented in Table 5. A p-value of less than 0.05indicates that the 2 sets of results being compared are statisticallydifferent from each other. Each of the ATP concentrations compared tobackground signal have a p-value of less than 0.05. Therefore, thisstatistical test indicates that each of the ATP concentrations analyzedis detectable over background.

TABLE 5 p-value 1.82 × 10⁻⁹M ATP 2.2 × 10⁻⁵ 9.1 × 10⁻¹⁰M ATP 9.5 × 10⁻⁸4.5 × 10⁻¹⁰M ATP 2.3 × 10⁻⁷

Example 3 Detection of dATP Using Luciferase

Detection of polydeoxyribonucleosides using Luciferase can in theory beperformed through the measurement of dATP if the enzyme used fordetection can utilize dATP. In this example, the ability of Luciferaseto use deoxyadenosine triphosphate (dATP) as compared to adenosinetriphosphate (ATP) was tested.

Reactions were assembled containing 50 μl of LAR, 2 or 4 μl ofluciferase stock (providing 5 or 10 ng of luciferase to the reactions)and 0 or 5 μl of 1 mM dATP Sigma (final concentration of dATPapproximately 100 μM). Luciferase was the last component added.Immediately upon enzyme addition, the light output of the reactions wasdetermined using a Turner TD-20e Luminometer. The results are providedin Table 6. These data show that Luciferase can be used to directlydetect dATP.

TABLE 6 Luciferase dATP Level + or − Light Units Avg.  5 ng −  423 295.7359.4  5 ng + 1450 1621 1535.5 10 ng −  703 705.5 704.3 10 ng + 36843441 3562.5

Example 4 Pyrophosphate Inhibition of Luciferase

The reaction of Luciferase produces pyrophosphate from ATP or dATP andis inhibited by pyrophosphate. Some of the reaction schemes describedlater use pyrophosphate as a substrate for other enzymes. In order touse levels of pyrophosphate in these reactions which do not inhibitdetection of nucleoside using Luciferase, we determined the levels ofinhibition produced by various concentrations of pyrophosphate on theproduction of light from Luciferase.

A new buffer, LAR without Coenzyme A, was made as described inExample 1. This buffer and the original LAR were then used to formulatevarious reactions with the compositions shown below. The reactions wereassembled with Luciferase being the final component added. Immediatelyupon enzyme addition, the light output of the reactions were determinedwith a Turner TD-20e Luminometer. The results are provided in Table 7.These data indicate that the light output from Luciferase can bemeasured in the presence of pyrophosphate and that more than 50% of theactivity can be seen with pyrophosphate concentrations as high as 100μM. In addition, these data indicate that removal of Coenzyme A from theLAR greatly lowers the background light produced by the reactionswithout greatly effecting the activity of Luciferase.

TABLE 7 LAR with LAR minus ATP pp_(i) Luciferase CoA (μl) CoA (μl) (2μM) 1 mM 100 μM (2.5 μg/ml) Light Units Avg. 50 μl — — − − 2 μl 209.9227 218.5 50 μl — 5 μl − − 2 μl 3462 3674 3568 50 μl — — + − 2 μl 9.739.54 9.6 50 μl — 5 μl + − 2 μl 169.5 180.9 175.2 50 μl — 5 μl − + 2 μl1452 1449 1450.5 — 50 μl — − − 2 μl 0.035 0.046 0.041 — 50 μl 5 μl − − 2μl 3735 3289 3512 — 50 μl — + − 2 μl 0.0003 0.0003 0.0003 — 50 μl 5 μl +− 2 μl 254.5 308 281.3 — 50 μl 5 μl − + 2 μl 2041 2069 2055

Example 5 Testing ADP as an Inhibitor of Luciferase

Some of the reaction schemes described later use adenosine diphosphate(ADP) as a substrate for other enzymes. ADP is a possible inhibitor ofLuciferase. Therefore, we determined the levels of inhibition producedby various concentrations of ADP on the production of light fromLuciferase and ATP.

Stock solutions of ADP Sigma or ATP were dissolved in 10 mM Tris-Cl pH7.5 and diluted to produce various stock concentrations. Reactions wereassembled which contained 2 μl of 2.5 μg/ml Luciferase, 50 μl of LAR, 5μl of ADP or 5 μl of 10 mM Tris Cl pH 7.5 and 5 μl of ATP or 5 μl of 10mM Tris Cl pH 7.5. The Luciferase was the final component added to thesereactions. Immediately upon enzyme addition, the light output of thereactions was measured using a Turner TD-20e Luminometer. The finalnucleoside concentrations of the reactions and the light output of thereactions are summarized in Table 8. These data indicate that ADP doesnot greatly effect the ability of Luciferase to produce light using ATPas a substrate. Thus, if low concentrations of ADP are added toLuciferase reactions from reactions performed using other enzymes,little effect on ATP detection through the use of Luciferase isexpected.

TABLE 8 ATP ADP Light output Average — — 485.8 423.5 454.7 2 μM — 49454930 4937.5 — 100 μM 4800 4418 4609 2 μM 100 μM 6834 7207 7020.5 —  1 μM513 463 488 2 μM  1 μM 4303 4152 4227.5 —  10 nM 419.6 419 419 2 μM  10nM 4534 4625 4579.5

Example 6 NDPK Transformation of ADP to ATP, Using Deoxynucleosides

Luciferase can detect ATP at much lower concentrations than dATP orother nucleosides. If deoxynucleoside triphosphates could be used togenerate ATP, an increase in sensitivity may result. For this reason, wetested the ability of enzymes to transfer the terminal phosphate ofdeoxynucleoside triphosphates to ADP, forming ATP and deoxynucleosidediphosphates.

Reactions were assembled which contained 100 μl of LAR, 10ng ofLuciferase in the presence or absence of deoxynucleoside triphosphates(1 μM final concentration when added), and 10 units of nucleosidediphosphate kinase (NDPK) (Sigma #N0379, Lot #127F81802). The reactionswere assembled with the exception of luciferase and incubated for 15 minat room temperature. The Luciferase was added and the light output ofthe reactions was measured immediately using a Turner TD-20eLuminometer. The light output values measured are provided in Table 9.These data confirm that NDPK is capable of transferring the phosphatefrom nucleoside triphosphates to ADP to form ATP which can be detectedusing Luciferase.

TABLE 9 Tube # dNTP ADP NDPK ATP Light Units 1 − + + 883 2 − − + + 153613 − + − 543 4 − − − + 21970 5 dATP + + 13356 6 dATP − + 151 7 dCTP + +13007 8 dCTP − + 6.9 9 dGTP + + 13190 10  dGTP − + 7.3 11  TTP + + 1923012  TTP − + 9.0

Example 7 NDPK Transformation of ADP to ATP Using NDPK and ATP Analogs

Some enzymes which may be used to transform nucleosides show specificityfor adenosine nucleosides as phosphate donors. Adenosine nucleosides maynot be used as high energy phosphate donors for these converting enzymesif a Luciferase detection system is to be utilized. This is becauselight would be generated by Luciferase from the added adenosinenucleoside. However, the converting enzymes may be utilized if an analogof adenosine is identified that can be used by the converting enzymesbut not by Luciferase. This example indicates how such analogs can betested for their ability to be used by converting enzymes but not byLuciferase.

Approximately 5 mg of ATP (Sigma A9187, Lot #36H7808), α,βmethyleneadenosine 5′-Triphosphate (AMP-CPP) (Sigma M6517, Lot #96H7813)and β,γ methylene adenosine 5′-triphosphate (AMP-PCP) (Sigma M7510, Lot#34H7840) were diluted in Tris-Cl, 10 mM, pH 7.5. The absorbance of a1:100 dilution of these solutions into 50 mM Tris-Cl, pH 7.5 was read at259 nm using a Beckman DU650 Spectrophotometer. The absorbances wereused to determine the concentration of these solutions using a molarextinction coefficient of 15.4×10³ M. Recombinant Luciferase was dilutedinto CCLR containing 1 mg/ml BSA to a concentration of 2.5 ng/μl. Whenthe reactions were assembled, 2 μl luciferase was added from the 2.5ng/μl stock solution and the light emission of the solutions wereimmediately read using a Turner TD-20e Luminometer. The data is providedin Table 10.

TABLE 10 Reaction LAR ATP AMP-CPP* AMP-PCP* # rxn Avg. 1 50 μl — — — 3426.4 2 50 μl 4 μM — — 7 5762 3 50 μl —  552 μM — 2 349.2 4 50 μl 4 μM 552 μM — 2 5072.5 5 50 μl — 5.52 μM — 2 465.8 6 50 μl 4 μM 5.52 μM — 25843.5 7 50 μl — 55.2 nM — 2 429.8 8 50 μl 4 μM 55.2 nM — 2 4152 9 50 μl— — 1.14 mM 2 260.35 10  50 μl 4 μM — 1.14 mM 2 3735.5 11  50 μl — —11.4 μM 2 431.25 12  50 μl 4 μM — 11.4 μM 2 5930 13  50 μl — —  114 nM 2389.35 14  50 μl 4 μM  114 nM 2 6093.5 *= final concentration in thereaction, solution produced by concentrated stock solution

Micromolar solutions of these ATP analogs do not produce light abovereactions containing no added nucleoside and do not greatly lower thelight output of reactions containing low levels of ATP from the valuesseen in the absence of these analogs. These analogs do not inhibitLuciferase and are not utilized by Luciferase. Thus, these data indicatethat these analogs can be tested for their ability to be used withenzymes for the transformation of nucleosides.

The following reactions were performed to determine if either AMP-CPP orAMP-PCP could be used by NDPK. All reactions were assembled in duplicateand incubated at room temperature for 20 min. Ten nanograms ofLuciferase was added and the light output of the reactions immediatelymeasured using a Turner TD-20e Luminometer. The data is provided inTable 11. These data demonstrate the analog AMP-CPP is utilized by theenzyme NDPK as a substrate to generate ATP from ADP. The values seenwith AMP-CPP, ADP and NDPK present are substantially higher than thoseseen for ADP alone, ADP and NDPK without AMP-CPP and NDPK alone.Analogous experiments can be performed to test other enzymes for theirability to use nucleoside substrates in a similar fashion.

TABLE 11 ADP AMP-CPP AMP-PCP Reac- LAR- (2 × (2 × (2 × tion CoA 10⁻⁴M)NDPK 10⁻⁵M) 10⁻⁵M) Avg. 1 100 μl — — — — 0.21 2 100 μl 0.5 μl — — —60.23 3 100 μl 0.5 μl 1 μl — — 59.77 4 100 μl 0.5 μl 1 μl 5 μl — 617.955 100 μl — 1 μl 5 μl — 1.81 6 100 μl 0.5 μl 1 μl — 5 μl 69.35 7 100 μl —1 μl — 5 μl 0.03 8 100 μl — 1 μl — — 0.05

Example 8 NMPK Transformations of ADP

This example demonstrates a method for testing the ability of an enzymeto transform nucleoside diphosphates into nucleosides which can be usedby Luciferase for the generation of light. The enzyme NucleosideMonophosphate Kinase (NMPK, Sigma, N-4379) can transfer the phosphatefrom ATP to UMP, forming UDP and ADP. This experiment demonstrates thatthis enzyme preparation can also be used to form ATP from ADP, probablythrough the reaction:

2ADP→ATP+AMP

The reactions were assembled in duplicate as prescribed in Table 12 andincubated at room temperature for 30 min. At that time, long ofLuciferase was added and the light output of the solutions was measuredusing a Turner TD-20e Luminometer. The data is provided in Table 12.These data indicate that NMPK can transform ADP into ATP. Similarexperiments can be used to test the ability of other enzymes to performsimilar transformations.

TABLE 12 Reaction LAR-CoA* NMPK** ADP (10⁻⁵M) Avg. Light 1 100 μl — —0.58 2 100 μl 10 μl — 0.23 3 100 μl — 5 μl 14.81 4 100 μl 10 μl 5 μl211.4 *= Luciferase Assay Reagent formulated without added Coenzyme A orATP **= Sigma N-4379, Lot #96H0166, dissolved in ATP free water to 3.35U/ml

Example 9 Combination of NMPK and NDPK, dCTP, and AMP

One potential method for amplifying an ATP signal requires two enzymesand a phosphate donor. For this system to operate, the first enzyme, E1,must be able to convert AMP to ADP but must be unable to use thephosphate donor. The second enzyme, E2, must be able to effectively usethe phosphate donor to transform any ADP formed to ATP. This exampledemonstrates a method to test the ability of a combination of enzymes tobe used in such a combination reaction scheme. The Examples abovedemonstrate that NDPK can transform ADP to ATP using dCTP as a phosphatedonor.

The reactions were assembled as presented in Table 13 and incubated for30 min at room temperature. Then 10ng of luciferase in 2 μl of 1×CCLRwith 1 mg/ml BSA was added and the light output of the reactions wasmeasured using a Turner TD-20e Luminometer. The data is presented inTable 13.

The reaction which could have produced significant ATP if NMPK couldtransform AMP to ADP using dCTP as a substrate is reaction 4. Thisreaction produced only minute amounts of ATP as measured by Luciferasemediated light production. Reaction 5 (where NDPK was used to transformADP to ATP using added dCTP) and reaction 6 (where NMPK was used totransform ADP to ATP) produced much more ATP. Since all enzymes wereshown to be active, these data indicate that NMPK essentially cannot usedCTP to transform AMP to ADP. This is the essential requirement for theATP amplification system described above. In this particular instanceE1, NMPK, cannot use the phosphate donor (dCTP) but can utilize AMP andATP to produce 2 ADP molecules (the reverse of the reaction 6). Thesecond enzyme, E2 (NDPK), can use the phosphate donor (dCTP) totransform the ADP produced by the first enzyme to create 2ATP from the2ADP using 2dCTP. These ATPs can then re-enter the cycle. This protocolcan be used to test combinations of enzymes and phosphate donors fortheir ability to act as the enzymes in our ATP amplification schemes.

TABLE 13 Reaction LAR-CoA AMP dCTP NMPK* NDPK** water Tris-Cl ADP Light1 100 μl — — 10 μl 1 μl — 15 μl  — 0.0088 2 100 μl 5 μl — — — 11 μl 10μl  — 0.126 3 100 μl — 10 μl — — 11 μl 5 μl — 0.159 4 100 μl 5 μl 10 μl10 μl 1 μl — — — 10.93 5 100 μl —  5 μl — 1 μl 10 μl — 10 μl 4049 6 100μl — — 10 μl  1 μl 5 μl 10 μl 2309 7 100 μl — — — — 11 μl 5 μl 10 μl115.9 * = NMPK concentration at 5 mg/ml of Sigma N4379, ** = NDPKconcentration of 10 units/μl Sigma N0379, Nucleoside stocks at: AMP(Sigma A2002, Lot #20H7035), 2 × 10⁻⁵ M; dCTP (Promega U122A, Lot#6858402), 2 × 10⁻⁵ M, ADP 1 × 10⁻⁵ M (Sigma A2754 Lot #65H7880)

Example 10 Amplification of ATP Using NMPK, NDPK, dCTP and AMP with ATPSpikes

The enzyme combination presented in Example 9 should be capable ofgreatly increasing the relative ATP concentration through the cyclicamplification reaction scheme presented earlier. This exampledemonstrates the amplification of different levels of input ATP usingthese enzymes and nucleosides. The reactions were assembled as presentedin Table 14 and incubated at room temperature. When the reactionsreached incubation time 0 min, 20 min, 40 min, 60 min, 80 min, 100 min,120 min, 180 min, and 240 min, 112 μl samples of each reaction weretransferred to luminometer tubes and 10 ng of luciferase were added. Thelight output of the reactions was immediately measured using a TurnerTD-20e Luminometer. The data is presented in Table 15.

The reactions with ATP added (Reactions 1, 2, and 3) increased in ATPmore rapidly than the reactions without added ATP (Reaction 4). The rateof increase of the ATP was dependent upon the amount of ATP first addedto the reaction. Thus, this combination of enzymes amplified the inputATP signal and the amount of ATP produced at a particular time wasdependent upon the starting amount of ATP added.

In addition, this combination of reactions allows the user to determineif any of the enzymes used are contaminated with unexpected activitiesthat may influence the system. For example, removing the NMPK, dCTP orAMP from the system prevents any ATP accumulation, as expected. However,eliminating the NDPK only has a small influence on the rate of ATPaccumulation. These data suggest that the NMPK source used contains asmall amount of activity which can take the place of NDPK in thissystem.

Performing similar experiments should allow a user to determine if otherenzymes can be used in such amplifications schemes, as shown in Example11.

TABLE 14 Reaction AMP dCTP NMPK NDPK ATP 1 10 μl 10 μl 100 μl 10 μl 1pmol 2 10 μl 10 μl 100 μl 10 μl 100 fmol  3 10 μl 10 μl 100 μl 10 μl 10fmol  4 10 μl 10 μl 100 μl 10 μl — 5 10 μl 10 μl — 10 μl 1 pmol 6 10 μl10 μl 100 μl — 1 pmol 7 — 10 μl 100 μl 10 μl 1 pmol 8 10 μl — 100 μl 10μl 1 pmol

TABLE 15 Reaction 0 min 20 min 40 min 60 min 80 min 100 min 120 min 180min 240 min 1 95 179.5 249.9 339.5 562 670.9 709.4 1157 1497 2 20.4237.11 54.95 85.1 122.5 164.9 206.1 409.7 746 3 12.3 25.09 39.37 55.7993.8 120.5 156.1 325.7 573.7 4 11.8 25.54 37.45 54.64 87.8 113.1 143.8299.1 548.7 5 96.4 75 63.63 56.75 51.72 54.72 56.61 58.97 60.97 6 110183.5 247.8 285.9 426.1 503.9 624.5 958 1301 7 91.7 99 98.6 85.2 93.294.2 95.4 91.5 90.4 8 3.521 2.755 2.31 2.058 2.092 2.173 1.682 1.0880.73

Example 11 Amplification of ATP Using Adenylate Kinase and PyruvateKinase

This example demonstrates a second ATP amplification system using anon-nucleoside based phosphate donor. The enzymes used are: adenylatekinase (an enzyme which produced 2ADP from one ATP and one AMP but whichcannot use phosphoenol pyruvate (PEP) as a phosphate donor and PyruvateKinase (an enzyme which phosphorylates ADP to form ATP using PEP as aphosphate donor). The reactions were assembled as presented in Table 16.These reactions were incubated at room temperature and 109 μl of thereactions was removed at 0, 30, 60, and 120 min. Luciferase (2 μl, 10 ngin 1×CCLR with 1 mg/ml BSA) was added and the light output of thereaction was immediately measured using a Turner TD-20e Luminometer. Thedata is presented in Table 17.

After 30 minutes of incubation the reaction containing ATP (reaction 1)increased much more rapidly than the reaction with no ATP added(reaction 2). Thus, the ATP sample was amplified. Also note that in thisset of reactions, the ATP content of reactions 1 and 2 reached a finalATP level. This indicates that the reactions had reach an equilibriumvalue.

Finally, note that the reaction with no added AMP also increased overtime. This suggests that one of the components was contaminated witheither AMP or ADP. Further experiments demonstrated that thecontaminating nucleoside was present in the pyruvate kinase solutionused in this study. The following example demonstrates a method forremoving this contaminating nucleoside.

TABLE 16 Reaction ATP AMP AK PEP PK Tris Buffer LAR-CoA 1 12.5 μl 5 μl10 μl 5 μl 12.5 μl — — 500 μl 2 — 5 μl 10 μl 5 μl 12.5 μl 12.5 μl — 500μl 3 12.5 μl — 10 μl 5 μl 12.5 μl   5 μl — 500 μl 4 12.5 μl 5 μl — 5 μl12.5 μl —   10 μl 500 μl 5 12.5 μl 5 μl 10 μl — 12.5 μl —   5 μl 550 μl6 12.5 μl 5 μl 10 μl 5 μl — — 12.5 μl 500 μl *The concentrations ofthese components were: ATP, 1 × 10⁻⁶ M; AMP, 1 × 10⁻⁴ M; AdenylateKinase (AK) (Sigma M5520, lot #16H9558), 7U/μl in 50 mM KPO₄, 15 mMMgCl₂, pH 7.5 (Buffer A); PEP, (Phosphoenolpyruvate, Sigma P-7002, Lot#46H3777, 100 mM in deionized water; PK (pyruvate kinase, (Sigma P-7286,Lot #45H9504), 0.1U/μl in Buffer A), and; Tris Cl, 10 mM Tris Cl, pH7.5.

TABLE 17 Time (Min) Reaction 0 30 60 120 1 93.6 536.4 683.8 670.4 214.98 120.6 594.8 639.3 3 105.5 219.4 321 384.7 4 112.5 97.2 98.8 94.1 583.1 16.84 16.03 15.02 6 90.6 21.61 22.79 21.2

Example 12 Removal of Interfering Substances in Pyruvate Kinase UsingDialysis

This example demonstrates methods for detecting contaminatingnucleosides in enzymes used in the various technologies discussed in theother examples and removing the contaminating material.

Additionally, another amplification scheme is described. This schemeutilizes: Adenylate Kinase (E1); NDPK (E2); AMP; and the ATP analogAMP-CPP as the high energy phosphate donor. If AMP is left out of thisreaction, no increase in an initial ATP signal should take place unlessone of the other materials is contaminated with AMP (or ADP).

Reactions performed as described in Example 11 suggested that one of thecomponents may have adenosine nucleoside contamination. One of thecomponents suspected of contamination is pyruvate kinase. A sample ofthis enzyme was dialyzed against 50 mM KPO₄, 15 mM MgCl₂ pH 7.6 inSpectra Por Dialysis tubing with a molecular weight cut off of 3,500 da.The dialysis was performed twice against 1000×amount of buffer forseveral hours at 4° C. to remove free adenosine. The reactions wereassembled according to Table 18. These data indicate that followingdialysis the enzyme solution was slightly more dilute than prior todialysis. By adding 5.3 μl of the post dialysis enzyme and 5.0 μl of thepre-dialysis enzyme, equal amounts of PK were added to the reactions.

500 microliters of LAR-CoA was added to the assembled reactions and thefinal reactions incubated at room temperature. At 0, 10, 20, and 30 min,114 μl of these reactions were added to 10 ng of luciferase in 5 μl of1×CCLR with 1 mg/ml BSA and the light output of the solutions wasimmediately measured using a Turner TD-20e Luminometer. The data ispresented in Table 19.

Two main observations can be derived from this data. First, the AK,NDPK, AMP, AMP-CPP enzyme-substrate combination can be used to amplifyan ATP signal. However, production of ATP from some contamination sourceallows reactions not given ATP added to achieve a final ATPconcentration similar to those given an ATP spike.

The reaction to which no AMP or PK were added (reaction 2) does notincrease over time. However, the reactions to which the undialized PKwas added and no AMP was added give high light output over time(reaction 5). Addition of dialyzed PK to reactions lacking AMP (reaction8) demonstrate increased light output over time, but the rate ofincrease is dramatically reduced from that seen without dialysis.

This Example demonstrates that yet another ATP amplification system canbe used to generate higher ATP levels from a starting ATP spike. Inaddition, this Example shows that these systems can be used to determineif solutions contain contaminating nucleosides and that dialysis can beused to fractionate contaminating nucleosides from enzymes utilized inATP amplification reactions.

TABLE 18 Reaction AMP ATP NDPK AMP-CPP AK PK Pi Buffer Tris 1 10 μl 10μl 5 μl 10 μl 10 μl — 5 μl — 2 — 10 μl 5 μl 10 μl 10 μl — 5 μl 10 μl 310 μl — 5 μl 10 μl 10 μl — 5 μl 10 μl 4 10 μl 10 μl 5 μl 10 μl 10 μl 25μl Sample 1 — — 5 — 10 μl 5 μl 10 μl 10 μl 25 μl Sample 1 — 10 μl 6 10μl — 5 μl 10 μl 10 μl 25 μl Sample 1 — 10 μl 7 10 μl 10 μl 5 μl 10 μl 10μl 26.5 μl Sample 2 — — 8 — 10 μl 5 μl 10 μl 10 μl 26.5 μl Sample 2 — 10μl 9 10 μl — 5 μl 10 μl 10 μl 26.5 μl Sample 2 — 10 μl The compositionsof these solutions were: AMP, 1 × 10⁻⁴ M; ATP, 2 × 10⁻⁶ M; NDPK,0.1U/μl; AMP-CPP, 1 × 10⁻³ M; AK, 0.75 units/μl; PK, 5 μl of pyruvatekinase pre-dialysis (sample 1) or post dialysis (sample 2), Pi buffer(described above); and, Tris, - Cl, pH 7.5.

TABLE 19 Time (Min) Reaction 0 10 20 30 1 60.35 349.7 529 563.1 2 54.7350.74 49.79 52.79 3 51.62 59.11 279 420.7 4 87.4 666.6 754.2 779 5 73.1213.9 354.6 412.4 6 24.41 479.1 701.7 707.6 7 69.9 449.3 577.3 595.2 850.92 76.03 118.4 148.9 9 12.92 229.3 541.3 569.4

Example 13 PRPP Synthetase, Reactions with Adenosine

The enzyme 5′ phopshorylribose 1′ pyrophosphate synthetase (PRPPSynthetase) transfers a pyrophosphate from ATP to D-ribose 5′ phosphate.This experiment was performed to determine if this enzyme could be usedwith AMP and 5′ phosphoribose 1′ pyrophosphate to generate ATP andD-ribose 5′ phosphate.

ATP, AMP and PRPP were diluted in 10 mM Tris, pH 7.3. PRPP Synthetase(Sigma #P0287) was diluted in PRPP Synthetase reaction buffer (seebelow). 2 μl of ATP, 2 μl of AMP, 2 μl of PRPP, and 2 μl of PRPPSynthetase (or appropriate buffers) were added as indicated in Table 20to 20 μl of PRPP Synthetase reaction buffer.

The reactions were incubated in the 37° C. water bath for 30 min. Thetubes were removed from the water bath and 100 μl of LAR (without CoA)was added. Then, 126 μl was transferred to a luminometer tube. 10 ng ofLuciferase was added in 5 μl of 1×CCLR containing 1 mg/ml BSA and lightoutput measured with Turner TD-20e Luminometer. The data presented inTable XXX. This data demonstrates that PRPP Synthetase can transferpyrophosphate from non-nucleoside substrates to AMP to form ATP.

The nucleoside concentrations in the reaction were: ATP (when added)1.2×10⁵M; AMP (when added) 2.9×10⁻⁵M, and; PRPP (when added) 2.6×10⁻⁵M.6×10⁻⁴ units of the enzyme (PRPP Synthetase) was added per reaction.PRPP Synthetase buffer is 50 mM triethanolamine, 50 mM potassiumphosphate, pH 7, 0.37 mM EDTA, 10 mM MgCl₂, 1 mg/ml BSA.

TABLE 20 Tube # AMP PRPP PRPP Syn Light Units 1 + + + 3440 3424 2 + − +0.522 0.501 3 + + − 6.649 4.619 4 − + + 7.096 7.139 5 + − − 1.874 0.4306 − − − 5.203 4.794 7 − + + 96.0 0.361 8 − − − 462.8 0.603

Example 14 PRPP Synthetase, Reactions with Deoxyadenosine

Some schemes for the detection of DNA require the conversion of dAMP,generated by nuclease digestion of DNA, to dATP. This exampledemonstrates that the enzyme PRPP Synthetase can perform thetransformation of dAMP to dATP using PRPP as a cosubstrate. In addition,this transformation can be monitored by Luciferase detection at muchhigher sensitivities if the dATP formed is used to transform ADP to ATPthrough the action of NDPK added to the reaction.

The reactions were assembled in duplicate as shown in Table 21. Theconcentrations of the reaction components were: dAMP 2.9×10⁻⁴ M in 10 mMTris pH 7.3; AMP 2.9×10⁻⁴ M in 10 mM Tris pH 7.3; PRPP 2.6×10⁻⁴ M in 10mM Tris pH 7.3; PRPP Syn (PRPP Synthetase) (Sigma #P0287) 100×dilutionof stock enzyme which is at 0.03 units/μl. The components were added totwenty microliters of PRPP Synthetase Buffer (see Example 13). Afterincubating for 47 min at 37° C., After incubating, 100 μl of LAR wasadded to all reactions along with 10 ng of luciferase and the lightoutput of the reactions was immediately measured. The data is presentedin Table 22. PRPP was able to utilize dAMP as a substrate (comparingreaction 1 to 2, 3, 4 and 5). However, the amount of light produced byreaction was not very great, probably due to the fact that luciferaseuses dATP at a much lower efficiency than ATP as presented earlier.

TABLE 21 Reaction dAMP PRPP PRPP Syn 1 2 μl 2 μl 2 μl 2 2 μl — 2 μl 3 2μl 2 μl — 4 2 μl — — 5 — 2 μl 2 μl

TABLE 22 Reaction Tube A Tube B Avg. 1 18.2 22.1 20.15 2 1.4 1.4 1.4 34.2 3.8 4 4 2.1 1.8 1.95 5 13.1 15.8 14.45

In order to demonstrate the transfer of phosphate from dATP to ADP toform ATP, the reactions presented in Table 23 were assembled induplicate in twenty microliters of PRPP Synthetase Buffer (for solutioncompositions, see tables above). They were then incubated at 37° C. for34 min. The added components had the following formulations: ADP2.3×10⁻² M in 10 mM Tris pH 7.3; NDPK-1000×dilution of Sigma #N0379 at10 units/μl (final concentration 0.01 units/μl). The tubes were thenincubated for an additional 60 min at 37° C., 10ng of Luciferase wereadded, and the light output was measured using a Turner TD-20eLuminometer. The data is presented in Table 24. These data indicate thatthe dATP produced by the PRPP Synthetase reaction can be transferred toADP by the action of NDPK to produce ATP.

TABLE 23 Reaction dAMP PRPP PRPP Syn ADP NDPK 1 2 μl 2 μl 2 μl 2 μl 2 μl2 2 μl 2 μl 2 μl — — 3 2 μl 2 μl 2 μl 2 μl— — 4 2 μl 2 μl 2 μl — 2 μl 5— 2 μl 2 μl 2 μl 2 μl

TABLE 24 Light Units Reaction Tube A Tube B 1 812.1 839.3 2 19.2 37.5 353.6 52.6 4 168.4 173.1 5 43.6 38.9

Example 15 Digestion of PolydA Using Nucleases

One potential method for detecting DNA would be to digest the polymer todeoxynucleoside 5′ monophosphate, transform the deoxynucleosidemonophosphates to deoxynucleoside triphosphates, form ATP from thedeoxynucleoside triphosphates using ADP and NDPK, and then detect theATP using Luciferase. This example demonstrates the digestion of adeoxyadenosine polymer.

A solution of deoxyadenosine 5′ monophosphate was made by adding 990 μlof water and 10 μl of 1×TE buffer (10 mM Tris Cl, 1 mM EDTA pH 8.0) to25 units of polyadenylic acid (Pharmacia 27-786, Lot #5017836021). Areaction was assembled with the following materials: 450 μl of nanopurewater, 50 μl of 10×S1 Nuclease buffer (Promega Corp. M577A, Lot#6748605) and 10 μl of the polydeoxyadenylic acid solution above. Theabsorbance change at 260 nm was monitored on a Beckman DU650Spectrophotometer. The rate of change in the absorbance of the solutionwas 0.0020 Abs/min. At this point, 1 μl of S1 Nuclease (Promega Corp.E576B, Lot #6800810) was added and the absorbance change of the solutionredetermined and found to be 0.0156 abs/min. Since smalloligonucleosides and mononucleosides display absorbance values higherthan a corresponding amount of polynucleoside, this indicates that thisenzyme can digest the polymer.

The reaction conditions given below are those used to digest thepolydeoxyadenylic acid polymer samples that are used in later examples.

Three reactions were assembled which contained:

Reaction 1: 90 μl of the polydeoxyadenylic acid solution describedabove, 10 μl of 10×S1 nuclease reaction buffer.

Reaction 2: As Reaction 1 above.

Reaction 3: 90 μl of nanopure water, 10 μl of S1 nuclease reactionbuffer.

At time equals zero minutes of digestion, 10 μl of each of these wasremoved and added to 490 μl of 50 mM Tris Cl pH 8.0. Immediately, 1 μlof S1 nuclease was added to the remaining reaction mixtures 1 and 3 butnot 2, and the mixtures were allowed to incubate at room temperature.Additional 10 μl samples of the reactions were removed after 20, 50 and140 min of reaction and diluted into 490 μl of 50 mM Tris Cl pH 8.0. Thedata is presented in Table 25. The absorbance of the solution inReaction #1 increased, again indicating that the polymer in thisreaction was digested over time. A second set of reactions was producedas described above. The only difference with these reactions was that 50units of Sigma Poly(dA)(Sigma P-0887, Lot #67H0226) was dissolved in 1.5ml of TE buffer and used in the reactions. After the 140 minutes ofdigestion, these reactions were used as described in Example 16.

TABLE 25 Net Absorbance at 260 nm of Samples From Reaction Time #1 #2 #30 0 0 0 20 0.0726 −0.0088 −0.0025 50 0.1425 0.0291 −0.0041 140 0.1445−0.003 −0.0044

Example 16 Detection of Poly (dA) Using Nucleases and PRPP Synthetase

In this example, the digested polynucleoside described in Example 15 isdetected by two different methods. Both methods begin withtransformation of the deoxynucleosides to deoxynucleoside triphosphatesusing PRPP Synthetase and PRPP. In the first method, ADP is converted toATP using the deoxynucleoside triphosphates formed in the PRPPsynthetase reaction and the resulting ATP detected using Luciferase. Inthe second method, AMP is converted to ATP using the deoxynucleosidetriphosphates formed by the PRPP Synthetase reaction, simultaneouslyamplified and detected using Luciferase.

Table 26 presents the components of the PRPP Synthetase reaction. Theconcentrations of the components were: PRPP, 2.6×10⁻⁴M in 10 mM Tris-ClpH 7.5; PRPP Synthetase, 6×10⁻⁴ Units of Sigma P0287 per 2 μl in PRPPSynthetase Buffer. For composition of Buffer, refer to PRPP SynthetaseBuffer in Example 13. The nucleoside digests containing S1 were dilutedin deionized water to yield the amount of polymer listed in the Table in8 μl of solution and added to the appropriate reactions. The digestcontaining no polymer was diluted identically to those with polymer.Eight microliters of this solution contained all the components in thesamples containing 720 ng of polymer except the Poly(dA). All thereactions were incubated 32 min in a 37° C. water bath. At this pointall the reactions were heated at 95° C. for 5 min to inactivate the PRPPSynthetase and cooled in an ice bath for 5 min.

TABLE 26 PRPP Reaction Digest Buffer PRPP Synthetase poly (dA) S1 1 720ng 80 μl 2 μl 2 μl — — 2 72 ng 80 μl 2 μl 2 μl — — 3 7.2 ng 80 μl 2 μl 2μl — — 4 0.72 ng 80 μl 2 μl 2 μl — — 5 — 80 μl 2 μl 2 μl 720 ng — 6 — 80μl 2 μl 2 μl — (720 ng

A. First Detection Method

Twenty microliters of each reaction was added to 100 μl of LAR minusCoA. Ten nanograms of Luciferase was immediately added and the lightproduction of the reactions was measured. A second 20 μl sample wasadded to 100 μl of LAR minus CoA, followed by addition of ADP (2 μl of 2μg/ml stock) and NDPK (2 μl of 1×10⁻² U/μl), and allowed to incubate 20min at room temperature. After the incubation, 10 ng of luciferase wasadded to the reactions and the light production of the reactions wasmeasured using a Turner TD-20/20 luminometer at 52.1% sensitivity. Thedata obtained for these measurements are presented in Table 27.

These data show that direct measurement of the deoxynucleosidetriphosphates is possible using luciferase if relatively high amounts ofdigested DNA are to be detected (see reaction 1 vs. 5 and 6 in the noNDPK column). However, much more sensitive detection is provided whenthe deoxynucleoside triphosphates are used to convert ADP to ATP usingNDPK.

TABLE 27 Light Units Reaction DNA no NDPK w/NDPK 1  180 ng 43 711 2   18ng 15 227 3  1.8 ng 13 77 4 0.18 ng 11 37 5 no S1 13 161 6 no poly 11 28

B. Second Detection Method

Twenty microliters of the reaction mixtures from the heat inactivatedPRPP Synthetase reactions were added to ATP amplification reactions inan attempt to use the initial deoxynucleoside triphosphates to produceof ATP. This would allow easier detection of the dATP produced by thePRPP synthetase reaction.

The reactions were assembled as demonstrated in Table 28. The reactionswere mixed and the first aliquot of 109.3 μl (1/7 of the reaction) wasremoved immediately after adenylate kinase was added. The aliquot wasplaced in a luminometer tube, 10 ng luciferase was added, the tubetapped to mix, and then the light output was measured with a TurnerTD-20/20 luminometer at 52.1% sensitivity. Subsequent aliquots wereremoved at 20 minute intervals and measured immediately. The reactionswere incubated at room temperature. The data obtained is presented inTable 29.

These results show that it is possible to amplify the dATP produced fromdigested DNA after conversion to nucleoside triphosphates. Note that thelight output obtained by this method is greater than the light output ofthe non-amplified PRPP Synthetase method.

TABLE 28 (Reaction Components**) Reaction DNA LAR-CoA AMP PEP AK PK TrisPRPP Buffer 1 poly(dA) 180 ng, 20 μl* + + + + + — — 2 poly(dA) 18 ng, 20μl* + + + + + — — 3 poly(dA) 1.8 ng, 20 μl* + + + + + — — 4poly(dA) + + + + + — — 5 poly(dA) no S1 + + + + + — — 6 S1 nuclease,no + + + + + — — 7 ATP 14 μl 2 mM + + + + + — 6 μl 8 dATP 14 μl 2mM + + + + + — 6 μl 9 dATP 14 μl 200 nM + + + + + — 6 μl 10 dATP 14 μl20 nM + + + + + — 6 μl 11 none + + + + + 14 μl 6 μl *These reactionsused 20 μl of the heat-inactivated PRPP Synthetase reactions from thefirst part of this example. **The components were: ATP (Sigma A9187) in10 mMTris pH7.5, dATP (Sigma D6500) in 10 mMTris pH7.5, AMP 7 μl of 2 ×10⁻⁴ M in 10 mM Tris pH 7.5, LAR-CoA (LAR without CoA) 700 μl perreaction tube, PEP (phosphoenol pyruvate-ammonium salt) (synthesized) 7μl of 100 mM, AK (adenylate kinase/myokinase) (Sigma M5520) 14 μl of0.75 units/μl in Buffer A, PK (pyruvate kinase) (Sigma P7286, dialyzed48 hours) 17.5 μl of 0.13 units/μl, Tris 10 mM pH 7.5, PRPP SynthetaseBuffer #-see example 13.

TABLE 29 0 minutes* 20 minutes* 40 minutes* 1 11.56 187.30 5860.0 2 1.8925.26 1598.0 3 2.09 12.52 671.3 4 1.34 20.21 1638.0 5 1.56 17.04 1009.06 1.11 9.44 691.1 7 27.21 315.30 7426.0 8 8.84 186.20 7177.0 9 1.52 9.76295.6 10 1.14 5.18 184.1 11 0.72 4.10 169.7 *Light output in RelativeLight Units

Example 17 Digestion of Phi×174 HinF1 Fragments

Polynucleoside encountered in nature is often double stranded. The DNAfragments generated by digestion of Phi×174 DNA using endonuclease HinFI are double stranded DNA fragments of various sizes. In order to testwhether double stranded DNA could be detected, the Phi×174 was directedused as a test substrate or digested with nucleases to producenucleosides which could be converted to nucleoside triphosphates as inprevious Examples.

The following conditions were used to digest DNA fragments frombacteriophage Phi×174. These materials were placed in three 1.5 mlpolypropylene tubes: 50 μl of Phi×174 Hin FI fragments (Promega G175A,Lot #773603); 40 μl of 5 mM Mg SO₄; 5 μl of Exo III buffer (10×)(Promega E577B, 4853216), and 5 μl of Nanopure water. Fifty microlitersof TE buffer and 40 μl of 5 mM MgSO₄; 5 μl of ExoIII buffer (10×) and 5μl of Nanopure water were added to one sample. Two of the samplescontaining Phi×174 DNA were further treated with 2 μl of Exo III(Promega M181A, 5512708) and the tubes placed in a 37° C. water bath for60 min. ExoIII was also deleted to the sample without DNA and the sampleincubated at 37° C. for 60 min.

At this time, 800 μl of nanopure water and 100 μl of S1 Nuclease Buffer(10×) (Promega M577A, Lot #6748605) were added to all samples. Threemicroliters of S1 nuclease (Promega E576B, Lot #789881) were then addedto all samples. All samples were incubated at 37° C. for 30 min.

Two hundred microliters from each of the three tubes containing DNA werediluted with 300 μl of 1×TE Buffer and the absorbance read at 260 nmusing a Beckman DU 650 spectrophotometer. The readings recorded were:tube one (no nuclease addition), 0.3073; tube two (treatment with ExoIII), 0.5495; tube three (treatment with Exo III and S1), 0.5190. Theincreased absorbance values of the tubes treated with nuclease indicatesthat the polymer was digested. These digests were subsequently used inother studies (see Example 18).

Example 18 Detection of Phi×174 Hin F1 Fragments Using Nucleases, PRPPSynthetase, NDPK

This example demonstrates the detection of DNA by digestion of thepolymer to nucleoside monophosphates using nucleases, transformation ofthe nucleoside monophosphates to nucleoside triphosphates using PRPPSynthetase and PRPP along with transformation of ADP to ATP using thenucleoside triphosphates generated by the action of PRPP Synthetase, anddetection of the ATP using Luciferase. A sample of deoxynucleoside (Poly(da)) was prepared as described in example 17. Different amounts ofdeoxynucleoside were used in the reactions as presented in Table 30.

The following additions were made to each reaction: 2 μl of PRPP, 2 μlof PRPP Synthetase, and 20 μl PRPP Synthetase buffer. The reactionsproceeded at 37° C. for 28 minutes at which time the reactions weretransferred to 100 μl LAR containing 2 μl of ADP and 2 μl of NDPK. Thissecond reaction was allowed to proceed at room temperature for 20 min.The amount of ATP produced was measured by the addition of 10 ng ofLuciferase followed by measuring light output with a luminometer. Thedata is presented in Table 30.

These data show that this combination of enzymes allows detection ofDNA.

TABLE 30 Light Reaction Nucleoside Amount in Rxn Units 1 dAMP 200 ng,600 pmoles 1018 2 dAMP  20 ng, 60 pmoles 636 3 dAMP  2 ng, 6 pmoles 1784 dAMP 200 pg, 600 fmoles 83 5 none  0 ng 69 6 Phi X174 only 100 ng 46(= 300 pmoles dNMP; approx. 75 pmoles dAMP) 7 Phi X174 + ExoIII 100 ng472 8 Phi X174 + Exo + S1  10 ng 448 9 No DNA + Exo + S1  0 ng 55

Example 19 Detection of Phi×174 Hin F1 Fragments Using ReverseTranscriptase and NDPK

The following example demonstrates the detection of DNA fragments havingnucleoside overhangs on their ends using reverse transcriptase. Thereactions were assembled as demonstrated in Table 31.

The components were: Buffer, 5×MMLV-RT Buffer, (Promega Part #M531A, Lot#7090101); DNA, Phi×174 Hin F1 Fragments (Promega Part #G175A, Lot#7733602); NaPP_(i), 10 mM Sodium Pyrophosphate (Promega Part #C113A,Lot#6675705); ADP, 1 μM ADP (Sigma A-5285, Lot #56H7815); NDPK,nucleoside diphosphate kinase, (Sigma N-0379, Lot #127F81802) 1 U/μl in25 mM sodium citrate; MMLV-RT (Promega M170A, Lot #6980019, 1 U/μl),incubated for 30 min at 37° C., then 2 μl of the reactions was added to100 μl of L/L (Promega FF2021, Enliten Luciferase/Luciferin Reagant).The light production by the reactions was immediately measured with aTurner TD-20e Luminometer. The data is presented in Table 31.

These data show that MMLV-RT can be used to pyrophosphorylate DNA andthat the resulting nucleosides can be used to transform ADP to ATP andthe ATP formed detected using Luciferase. Other enzymes can be testedfor their ability to perform this reaction in a similar fashion.

TABLE 31 Rx Buffer DNA NaPPi ADP NDPK water MMLV-PT Light 1 4 μl 1 μl of100 ng/μl 1 μl 2 μl 1 μl 10 μl 1 μl 165. 2 4 μl 1 μl of 20 ng/μl 1 μl 2μl 1 μl 10 μl 1 μl 155. 3 4 μl 1 μl of 4 ng/μl 1 μl 2 μl 1 μl 10 μl 1 μl58.9 4 4 μl 1 μl of 800 pg/μl 1 μl 2 μl 1 μl 10 μl 1 μl 18.0 5 4 μl 1 μlof 160 ng/μl 1 μl 2 μl 1 μl 10 μl 1 μl 4.54 6 4 μl 1 μl of 32 ng/μl 1 μl2 μl 1 μl 10 μl 1 μl 1.70 7 4 μl — 1 μl 2 μl 1 μl 11 μl 1 μl 0.95 8 4 μl1 μl of 1 μl 2 μl  1 μl 11 μl  0.62

Example 20 Limit of DNA Fragment Detection Using Reverse Transcriptase,NDPK, and Luciferase

As shown in Example 18, DNA can be detected using luciferase when theDNA is fragmented and pyrophosphorylated using a reverse transcriptaseto produce dNTPs and the terminal phosphate is transferred from thedNTPs to ADPs to form ATP. This Example demonstrates that the lightunits produced in reaction containing very low levels of DNA arestatistically significant compared to values for the appropriate controlreactions. As with Example 2, the limit of detection is statisticallydetermined using Student's t-Test. The reactions presented in Table 32were assembled in duplicate. The components were: Buffer 5×MMLV-RTBuffer (Promega M531A); DNA, Phi×174 Hin F1 fragments diluted in TE(Promega G175A); TE, Tris EDTA (Promega AA641); NaPPi, 40 mM SodiumPyrophosphate (Promega C113A); ADP, ADP 2 uM in Tris 10 mM pH 7.3 (SigmaA5285); NDPK, 1 unit/μl in water (Sigma N0379); water, nanopure water;MMLV-RT, MMLV reverse transcriptase 200 units/μl (Promega M170A).

All reagents except DNA and MMLV-RT were added to a 1.5 ml polypropylenetube and mixed. Then duplicate 16.5 μl aliquots were transferred to newpolypropylene tubes. One microliter of MMLV-RT was added to each tube,followed by 2.5 μl of DNA at varying concentrations or 2.5 μl of TE. Thereactions were incubated at 37° C. for 10 min, then 2 μl of the 20 μlreaction was added to 100 μl of L/L reagant (which includes luciferase,Promega F202A and F180A, mixed) in a luminometer tube. The tubes weretapped to mix, and then light output levels were immediately measuredusing a Turner TD-20e luminometer at 52.1% sensitivity (this sensitivityis comparable to the Turner TD-20/20 readings). The data is presented inTable 33.

The light output for each DNA concentration (6 readings each) wascompared to the light output of the background control (no DNA), and ap-value determined for each comparison. The results of the analysis arepresented in the following Table 34. As in Experiment 2, the p-valueswere less than 0.05 for each sample tested. Therefore, less than 10 pgof DNA can be reliably detected.

TABLE 32 Reaction Buffer DNA TE NaPPi ADP NDP water MMLV-RT 1 4 μl — 2.5μl 0.5 μl 1 μl 1 μl 10 μl 1 μl 2 4 μl 2.5 μl or — 0.5 μl 1 μl 1 μl 10 μl1 μl 40 pg/μl 3 4 μl 2.5 μl of — 0.5 μl 1 μl 1 μl 10 μl 1 μl 100 pg/μl 44 μl 2.5 μl of — 0.5 μl 1 μl 1 μl 10 μl 1 μl 200 pg/μl

TABLE 33 Reaction Amount of DNA Light Units 1 no DNA tube 1A 1.166 2 noDNA tube 1A 1.189 3 no DNA tube 1A 1.190 4 no DNA tube 1B 1.071 5 no DNAtube 1B 1.124 6 no DNA tube 1B 1.159 7 10 pg DNA tube 2A 1.355 8 10 pgDNA tube 2A 1.498 9 10 pg DNA tube 2A 1.464 10 10 pg DNA tube 2B 1.48511 10 pg DNA tube 2B 1.519 12 10 pg DNA tube 2B 1.189 13 25 pg DNA tube3A 2.360 14 25 pg DNA tube 3A 2.159 15 25 pg DNA tube 3A 2.344 16 25 pgDNA tube 3B 2.126 17 25 pg DNA tube 3B 2.087 18 25 pg DNA tube 3B 2.14819 50 pg DNA tube 4A 4.501 20 50 pg DNA tube 4A 4.920 21 50 pg DNA tube4A 4.751 22 50 pg DNA tube 4B 4.721 23 50 pg DNA tube 4B 4.809 24 50 pgDNA tube 4B 4.929

TABLE 34 Student's t-Test for DNA detection p-value 10 pg 0.002377925 25pg 3.9211E-07 50 pg 4.2734E-09

Example 21 Detection of Blunt End DNA Fragments Using ReverseTranscriptase and NDPK

The following example demonstrates the detection of DNA fragments havingblunt ends using reverse transcriptase. A reaction master mix was madecontaining: 80 μl of 5×MMLV-RT Buffer (Promega Part #M531A, Lot#7090101); 10 μl of 40 mM Sodium Pyrophosphate (Promega Part #C113A, Lot#6675705); 10 μl of 1 μM ADP (Sigma A-5285, Lot #56H7815); 20 μl of NDPK(Sigma N-0379, Lot #127F81802 1 U/μl); and 210 μl of deionized water.

DNA samples consisted of ladders of DNA fragments in multiples of 25 bp(Promega G451, Lot #84791) and 50 bp (Promega G452, Lot #84796) in 1×TEbuffer. These materials were diluted into 1×TE buffer to produce aseries of solutions at different DNA concentrations. The reactions wereassembled as demonstrated in Table 35. The composition of thesecomponents was: MM, Master Mix (described above), and 200 U/μl MMLV-RT(Promega Part #M531A).

These reactions were incubated for 30 min at 37° C. After incubation, 2μl of the solution was added to 100 μl of L/L reagent and the lightproduction of the reaction was measured using a Turner TD-20eLuminometer. The data is presented in Table 35. These data demonstratethat sensitive DNA detection of blunt ended fragments can be achievedthrough pyrophosphorolysis of the DNA followed by conversion of ADP toATP.

TABLE 35 Rx MM DNA MMLV-RT Light 1 18 μl 100 ng 25 bp ladder 1 μl 142.52 18 μl  20 ng 25 bp ladder 1 μl 66.28 3 18 μl  4 ng 25 bp ladder 1 μl20.33 4 18 μl 800 pg 25 bp ladder 1 μl 5.216 5 18 μl 160 pg 25 bp ladder1 μl 1.606 6 18 μl  32 pg 25 bp ladder 1 μl 0.902 7 18 μl — 1 μl 0.717 818 μl 100 ng 25 bp ladder — 0.571 9 18 μl 100 ng 50 bp ladder 1 μl 149.210 18 μl  20 ng 50 bp ladder 1 μl 84.43 11 18 μl  4 ng 50 bp ladder 1 μl27.56 12 18 μl 800 pg 50 bp ladder 1 μl 6.694 13 18 μl 160 pg 50 bpledder 1 μl 2.829 14 18 μl  32 pg 50 bp ladder 1 μl 1.323 15 18 μl — 1μl 0.951 16 18 μl 100 ng 50 bp ladder — 0.751

Example 22 Detection of PolyA RNA Using Poly A Polymerase

This example demonstrates the detection Poly A mRNA by thepyrophosphorylation of the poly A segment. The reactions were assembledas demonstrated in Table 36. The compositions of the reaction materialswas: 10×Buffer-0.5M Tris-HCl, pH 7.5, 0.1M MgCl₂, 0.5M NaCl; Globin mRNAGibcoBRL cat#18103-028 (dissolved in H₂O); NaPP_(I), 20 mM sodiumpyrophosphate (Promega C113A, in deionized water); Poly A Polymerase,(Sigma P4058, 1 U/μl). These reactions were incubated at 37° C. for 30min, then 2 μl of the reaction was added to 100 μl of L/L Reagent andthe light output of the reaction immediately measured using a TurnerTD-20e Luminometer. The data is presented in Table 37. These datademonstrate that Poly A Polymerase is capable of pyrophosphorylating theRNA and that the resulting nucleoside triphosphates can be detectedusing luciferase, even if only very low levels of RNA are present.

TABLE 36 Reaction 10X Globin NaPPi Poly A Water 1 2 μl 1 μl of 50 ng/μl1 μl 1 μl 15 μl 2 2 μl 1 μl of 10 ng/μl 1 μl 1 μl 15 μl 3 2 μl 1 μl of 2ng/μl 1 μl 1 μl 15 μl 4 2 μl 1 μl of 400 pg/μl 1 μl 1 μl 15 μl 5 2 μl 1μl of 80 pg/μl 1 μl 1 μl 15 μl 6 2 μl 1 μl of 16 pg/μl 1 μl 1 μl 15 μl 72 μl — 1 μl 1 μl 15 μl

TABLE 37 Reaction Light RNA Present in L/L 1 772.2 5000 pg 2 172.3 1000pg 3 33.53 200 pg 4 7.727 40 pg 5 1.85 8 pg 6 0.743 1.6 pg 7 0.5941

Example 23 Detection of Poly A RNA Using Reverse Transcriptase and NDPK

This example demonstrates another method for the detection of mRNA,particularly polyA mRNA. In this method, a DNA segment is hybridized tothe mRNA and the probe is pyrophosphorylated using a reversetranscriptase and pyrophosphate. As the pyrophosphorylation occurs, thedeoxynucleoside triphosphates are used to convert ADP to ATP using theenzyme NDPK. The ATP of the final solution is then measured usingluciferase.

The reactions were assembled as presented in Table 38. The reactioncomponents were: Buffer, 5×MMLV-RT Buffer (Promega Part #M531A, Lot#7090101); mRNA, Globin mRNA (GibcoBRL cat# 18103-028 dissolved in H₂O);Poly (dT), 0.2 μM oligo dT(50), NaPPi, 20 mM Sodium Pyrophosphate,(Promega C113A in deionized water); ADP, 10 mM ADP (Sigma A-5285 Lot#56H7815); NDKP, nucleoside diphosphate kinase, 1 U/μl, (Sigma N-0379Lot #127F81802); MMLV-RT, (Promega Part #M531A, Lot #7090101) 200 U/μl;and 200 U/μl Superscript II GibcoBRL cat# 18064-014).

These reactions were incubated at 37° C. for 30 min and 2 μl of thereactions was added to 100 μl of L/L reagent. The light production ofthe reactions was immediately measured using a Turner TD-20eLuminometer. The data is presented in Table 39.

TABLE 38 Rx Buffer mRNA Poly (dT) NaPPi ADP NDPK MMLV-RT Superscriptwater 1 4 μl 1 μl of 50 ng/μl 1 μl 1 μl 2 μl 1 μl 1 μl — 9 μl 2 4 μl 1μl of 10 ng/μl 1 μl 1 μl 2 μl 1 μl 1 μl — 9 μl 3 4 μl 1 μl of 2 ng/μl 1μl 1 μl 2 μl 1 μl 1 μl — 9 μl 4 4 μl 1 μl of 400 pg/μl 1 μl 1 μl 2 μl 1μl 1 μl — 9 μl 5 4 μl 1 μl of 80 pg/μl 1 μl 1 μl 2 μl 1 μl 1 μl — 9 μl 64 μl — 1 μl 1 μl 2 μl 1 μl 1 μl — 9 μl 7 4 μl 1 μl of 50 ng/μl 1 μl 1 μl2 μl 1 μl — 1 μl 9 μl 8 4 μl 1 μl of 10 ng/μl 1 μl 1 μl 2 μl 1 μl — 1 μl9 μl 9 4 μl 1 μl of 2 ng/μl 1 μl 1 μl 2 μl 1 μl — 1 μl 9 μl 10 4 μl 1 μlof 400 pg/μl 1 μl 1 μl 2 μl 1 μl — l μl 9 μl 11 4 μl 1 μl of 80 pg/μl 1μl 1 μl 2 μl 1 μl — 1 μl 9 μl 12 4 μl — 1 μl 1 μl 2 μl 1 μl — 1 μl 9 μl

TABLE 39 Rx mRNA Light Units 1 5 ng 647.2 2 1 ng 425.4 3 0.2 ng 113.9 440 pg 43.56 5 8 pg 23.66 6 — 21.52 7 5 ng 648.5 8 1 ng 500.4 9 0.2 ng144.2 10  40 pg 45.85 11  8 pg 28.17 12  — 19.71

Example 24 Detection of RNA Using Nucleases, PRPP Synthetase

This example demonstrates the detection of RNA by digestion of RNA bynucleases, transformation of the AMP produced to ATP by PRPP Synthetase,and detection of the ATP produced using Luciferase. Three reactions wereassembled. These were: Digest 1 (250 ng Globin mRNA and S1 in a 10 μlreaction); Digest 2 (same as Digest 1, however no S1 nuclease wasadded); Digest 3 (same as Digest 1, but without Globin mRNA). Afterthese digests had incubated 30 min at 37° C., they were used to composethe reactions presented in Table 40. [For the concentrations of thesesolutions, see descriptions under reaction composition table in Example16.]

The reactions were incubated for 30 min at 37° C., 100 μl of LAR-CoA and10 ng of Luciferase were added to each tube, and the light output of thereactions were measured using a Turner TD-20e Luminometer. The data ispresented in Table 40. These data show that this combination of enzymescan be used to detect relatively low levels of RNA.

TABLE 40 PRPP Digest Polymer Synthetase PRPP Light Reaction AMP # and μlAdded Buffer Synt Units 1 2 μl of — — 20 μl 2 μl 3869 2.9e-4M (200 ng) 22 μl of — — 20 μl 2 μl 1287 2.9e-5M (20 ng) 3 2 μl of — — 20 μl 2 μl192.9 2.9e-6M (2 ng) 4 2 μl of — — 20 μl 2 μl 118.6 2.9e-7M (200 pg) 5 —1, 0.2 5 ng 20 μl 2 μl 48.4 μl 6 — 1, 0.02 0.5 ng 20 μl 2 μl 14.8 μl 7 —2, 0.2 5 ng 20 μl 2 μl 10.9 μl 8 — 2, 0.02 0.5 ng 20 μl 2 μl 10.6 μl 9 —3, 0.2 — 20 μl 2 μl 10.3 μl 10  — 3, 0.02 — 20 μl 2 μl 11

Example 25 Improved Detection of Cells Through the Addition of Materialsthat Allow ATP to be Produced from the Enzymes in the Cells.

One common way to detect the presence of cells is to assay the ATPcontent of materials which may contain cells. However, such detectionmethods are limited by the very small concentration of ATP that ispresent in samples which may contain very few cells. Several types ofenzymatic activities are required in every living cell. These activitiesare involved in the transformation of nucleosides into nucleosidetriphosphates for use in cellular metabolism. In particular, theactivities known as adenylate kinase and nucleoside diphosphokinase arewidely found in cells. Since these enzymes are expected to exist in allcell lysates, addition of AMP and dCTP should result in the formation ofATP in cell lysates through the reactions:

AMP+dCTP+adenylate kinase→ADP+dCDP

ADP+dCTP+NDPK→ATP+dCDP

If, however, the enzymes which remove ATP from such extracts are activeenough to remove the ATP as it is formed, no build-up of ATP willresult.

This example demonstrates that ATP can be detected in cell lysatesamples to which AMP and dCTP are added. The nucleoside transformationssuch as those presented above probably increase ATP concentration.Therefore, lower amounts of cells and cellular materials can be detectedby taking advantage of the transformation activity of these enzymes toproduce ATP from AMP thereby detecting ATP directly.

A sample of E. coli JM109 was grown in Luria broth for 2 hours. Thecells were harvested by centrifugation at 7240 g for 10 min and thenresuspended in 1×TBS. The cells were spun down again the same way andresuspended in 1×TBS. A sample of the resuspended cells was removed,diluted into sterile Luria broth and plated onto Luria Agar plates todetermine the number of cells per microliter of resuspended cellculture. The cell culture was then lysed by sonication and the lysatewas used in the reactions presented in Table 41. After an incubation of40 min at room temperature, 10 ng of luciferase was added to thereactions and the light output of the reactions was immediately measuredusing a Turner TD 20-e luminometer. The data is presented in Table 41.

TABLE 41 AMP Reaction Lysate (0.1 mM) dCTP(1 mM) Buffer* Light 1 10 μl**— 2 μl 100 μl 142 2 10 μl** 2 μl — 100 μl 69 3 10 μl** — — 100 μl 67 4 —2 μl 2 μl 100 μl 4 5 10 μl** 2 μl 2 μl 100 μl 175 6 1 μl** 2 μl 2 μl 100μl 18.85 7 0.1 μl 2 μl 2 μl 100 μl 4.6 8 0.01 μl 2 μl 2 μl 100 μl 3.5*buffer = 100 μl LAR **Lysate made from resuspended cells at aconcentration of 1.8 × 10⁵ cells/μl. Dilutions of this lysate were madein 1 X TBS and 10 μl added to 5, 6, 7, and 8, the amount of initiallysate equivalent to that added is above.

This data shows that addition of dCTP (reaction 1) allows more light tobe measured from a cell sample than can be measured if no addition wasmade to the lysate (reaction 3) or if AMP alone was added to the lysate(reaction 2). However, even more light can be found if both AMP and dCTPis added to the lysate (reaction 5). Dilution of the lysate results in areduction of the light produced by reactions given AMP and dCTP(reactions 6 and 7). However, the amount of light found is still abovethe amount expected to present by simple dilution of the lysate. Thesedata then, show that improved cell detection can be demonstrated ifadditions can be made to the lysate which should result in an increasedATP level in the sample.

Example 26 Optimization of the Concentrations of Nucleotides Added toForm Additional ATP in Cell Lysates

Example 25 demonstrates that additional ATP can be detected in celllysates if materials are added to the lysate. This example demonstratesthat the concentration of the material added can be adjusted to producea substantially higher level of ATP than is originally present in thelysates. A cell lysate produced from a known amount of cells wasproduced as in Example 23. This new lysate was used in the reactionspresented in Table 42. The reactions were incubated for 40 min at roomtemperature. After incubation, 10ng of luciferase was added to thereactions and the light output of the reactions was immediately readusing a Turner TD-20e luminometer. The data is presented in Table 42.

These data indicate that the concentration of both nucleotides can beco-optimized to obtain light values far superior to those seen in eitherthe lysate without additions or with non-optimal additions.

TABLE 42 Stock AMP Stock dCTP Reaction conc. conc. Lysate LAR-CoA Light 1 — 100 mM 10 μl* 100 μl 144.8  2 80 mM — 10 μl* 100 μl 1.515  3 — — 10μl* 100 μl 38.4  4 80 mM 100 mM — 100 μl 1.743  5 0.1 mM   1 mM 10 μl*100 μl 139.4  6  1 mM  1 mM 10 μl* 100 μl 247.3  7 10 mM  1 mM 10 μl*100 μl 569.2  8 80 mM  1 mM 10 μl* 100 μl 336.7  9 80 mM  1 mM 10 μl*100 μl 273.9 10  1 mM  1 mM 10 μl* 100 μl 283.3 11  1 mM  10 mM 10 μl*100 μl 239.6 12  1 mM 100 mM 10 μl* 100 μl 358.8 13 10 mM  1 mM 10 μl*100 μl 666.7 14 10 mM  10 mM 10 μl* 100 μl 1236 15 10 mM 100 mM 10 μl*100 μl 2320 16 80 mM  1 mM 10 μl* 100 μl 339.8 17 80 mM  10 mM 10 μl*100 μl 761.9 18 80 mM 100 mM 10 μl* 100 μl 1970 *lysate was made fromresuspended cells at a concentration of 3 × 10⁴ cells/ul **2 μl of stockused

Example 27 Time Course of ATP Increase in Lysates Following Addition ofEnzyme Substrates

The examples above indicate cell detection sensitivity increased byaddition of dCTP and/or AMP followed by an incubation period prior toATP detection using luciferase. This example demonstrates that detectionreaction may be temporally optimized as well.

The cell lysate was made as in Example 25 and was frozen. This lysatewas thawed and used to compose the reactions presented in Table 43.Samples were removed from reaction 5 at 1, 5, 15, 30, 60, 90, 120, 150,and 180 min and at 5 min for the other reactions. These samples wereadded to 10 ng of luciferase and the light output of the reaction wasmeasured immediately using a Turner TD-20e luminometer. The results fromthe samples taken at 5 min are presented in Table 43. The results fromthe sample of reaction 5 taken over time are presented in Table 44.

Note that the light output for the reaction rises dramatically over timeand reaches final values far above those reported in the previousexample. These data indicate that the most sensitive detection of cellswill require optimization of the reaction time used for detection inaddition to optimization of the added materials.

TABLE 43 Reaction Lysate AMP dCTP Light 1 10 μl — 100 mM 329 2 10 μl 10mM — 80.4 3 10 μl — — 241.8 4 — 10 mM 100 mM 1.129 5 10 μl 10 mM 100 mM347.5

TABLE 44 Time Light  1 136.5  5 347.5  15 1325  30 6379  60 21078  9033204 120 41470 150 43844 180 36579

Example 28 Determination of the Effect of Increasing the Number of DNAEnds on Detection of DNA Through Pyrophosphorylation.

Reverse transcriptases and DNA polymerases usually bind to DNA segmentswhich can be used as substrates in polymerization reactions. PlasmidDNAs have no DNA ends since they are a covalently closed circularmolecule. In general, such a molecule would not be expected to undergopyrophosphorylation unless the DNA is first modified to transform itinto a substrate for the reverse transcriptase or polymerase. In thisexample, an experiment is described that confirms that plasmid DNA isnot as good a substrate for pyrophosphorylation as digested fragments.In addition, using an enzyme to cleave the DNA which generates more newDNA ends than one that generates fewer ends may improve the detection ofthe DNA.

The reactions were assembled as presented in Table 45. The componentswere: Plasmid, pGEM 3ZF(+) (1 mg/ml, Promega corporation, Part #P227A);Buffer, 10×Buffer B (Promega Corporation, Part #R002A); Sau 3AI,Endonuclease Sau 3AI, (Promega Corporation, 8 U/μl, Part #R619E); BamH1, Endonuclease Bam H1 (Promega Corporation, 10 U/μl, Part #R602A). Thesolutions were incubated at 37° C. for 1 hr, then heated at 70° C. for10 min, and allowed to cool to room temperature. The solutions were thenadded to the reactions presented in Table 46. The reactions wereincubated at 37° C. for 20 min. After incubation, 2 μl of the reactionsolution was added to 100 μl of L/L reagent and the light output of thereactions was immediately measured using a Turner TD-20e luminometer.The data is presented in Table 46.

These data again demonstrate that detection of DNA bypyrophosphorylation is possible. In addition, these data demonstratethat digestion of plasmid DNA is needed prior to treatment using reversetranscriptase. Bam H1 produces only one DNA fragment from the plasmidwhile Sau 3A produces over 10 fragments from this plasmid. These datademonstrate that light production increases with increasing fragment endnumber.

TABLE 45 Solution Plasmid Buffer Water Sau 3A Bam H1 1 1 μl 5 μl 44 μl —— 2 — 5 μl 45 μl — — 3 — 5 μl 44 μl — 1 μl 4 — 5 μl 44 μl 1 μl — 5 1 μl5 μl 43 μl — 1 μl 6 1 μl 5 μl 43 μl 1 μl —

TABLE 46 Reaction MM MMLV-RT Solution Light R × 1− 18 μl — 1 μl #1 0.87R × 1+ 18 μl 1 μl 1 μl #1 0.787 R × 2− 18 μl — 1 μl #2 0.906 R × 2+ 18μl 1 μl 1 μl #2 0.75 R × 3− 18 μl — 1 μl #3 0.932 R × 3+ 18 μl 1 μl 1 μl#3 0.714 R × 4− 18 μl — 1 μl #4 0.856 R × 4+ 18 μl 1 μl 1 μl #4 0.713 R× 5− 18 μl — 1 μl #5 0.837 R × 5+ 18 μl 1 μl 1 μl #5 2.909 R × 6− 18 μl— 1 μl #6 0.811 R × 6+ 18 μl 1 μl 1 μl #6 8.757

Example 29 Demonstration of DNA Detection Using PyrophosphorylationCatalyzed by a Thermostable DNA Polymerase.

Both reverse transcriptases and DNA polymerases catalyze the addition ofnucleotides to a DNA strand. As shown in the earlier examples, reversetranscriptases can be used to catalyze the pyrophosphorylation of DNAthereby allowing its detection using coupled enzymatic reactions. Inthis example, we demonstrate that DNA polymerases also can be used tocatalyze this reaction and that the DNA polymerase from Thermusaquaticus (Taq) in fact produces more light from a set amount of inputDNA than does the reverse transcriptase.

A master mix (MM) was made which comprised: 10×buffer (Promega Part#M190G, Lot #7675526), 20 μl, 25 mM MgCl₂, 40 μl , 40 mM SodiumPyrophosphate, 5 μl; Taq DNA Polymerase (Promega Part #M166B, Lot#7474623) [storage buffer b], 5 U/μl, 10 μl; water, 100 μl. Thissolution was mixed by vortex and then used to compose the followingreactions: Reactions 1-3 (17.5 μl of master mix, 2.5 μl of 1×TE);Reactions 4-6 (17.5 μl of master mix, 1 μl of 100 pg DNA/μl [Phi×174HinF1 Fragments, Promega G175A diluted to the concentration listed using1×TE buffer], 1.5 μl of 1×TE); and Reactions 7-9 (17.5 μl of master mix,2.5 μl of 100 pg DNA/μl). The solutions were mixed and 30 μl of mineraloil was used to cover the aqueous solution. The solutions were incubatedat 70° C. for 30 min. Fifteen microliters were removed to which 1 μl of1 U/μl NDPK and 1.5 μl of 1 uM ADP were added. After an additional 15min at room temperature, 2.3 μl of each sample was added to 100 μl ofL/L reagent. The light output of the reactions were immediately measuredusing a luminometer. The data is presented in Table 47.

These results demonstrate the pyrophosphorylation reaction can becatalyzed by DNA polymerases and that low amounts of DNA may bedetected. The values obtained from reactions with 10 and 25 pg of DNAare statistically different from the no DNA addition values.

Table 47 Reac- p- tions DNA* Light Units Measured Mean Sd. value** 1-3 0 pg 0.915 0.653 0.837 0.802 0.135 4-6 10 pg 5.718 7.718 7.397 6.9581.089 <.0094 7-9 25 pg 11.8 11.18 14.79 12.59 1.93 <.0086 *amount of DNApresent in the luciferase assay tube **p value for comparison of theresults from no DNA addition to this Mentioned in earlier examples, anyvalue <0.05 is usually considered a significant difference

Example 30 Additional DNA Detection Experiments

This example is a direct comparison of the detection of DNA by a reversetranscriptase (MMLV-RT) a thermostable DNA polymerase (Taq Polymerase)and a non-thermostable DNA Polymerase (T4 DNA Polymerase). Also shown isanother example of how the particular structure of the DNA fragmentsutilized in the reaction must be matched to the properties of the DNAmodifying enzyme. The enzymes generally fail to produce a signal fromsupercoiled plasmid DNA since all these enzymes require a DNA end tostart their reactions. MMLV-RT and Taq DNA Polymerase utilize DNAspecies having a 5′ overhang but cannot use a DNA having a 3′ overhangas a substrate. In contrast, T4 DNA Polymerase utilizes DNA substrateswith both 5′ overhangs and 3′ overhangs. This ability may be due to its3′ exonuclease activity. In addition, this Example shows that reactionsusing T4 DNA polymerase produce more light than from equivalentreactions with either of the other two enzymes.

The reactions were assembled as presented in Table 48. The solutionswere incubated at 37° C. for 1 hr then at 70° C. for 10 min. At thispoint, 1 μl of each reaction was diluted to 20 μl with water to give aconcentration of 100 Opg DNA/μl. Solution MM was made as follows: 40 μl5×MMLV-RT Reaction Buffer (Promega Part M531A); 5 μl 40 mM Sodiumpyrophosphate; 20 μl 1 μM ADP; 5 μl 1 U/μl NDPK; and 180 μl water. Thereactions were mixed and 18 μl were transferred into 8 tubes. Onemicroliter of reaction 1 above and 1 μl of MMLV-RT (200 U/μl ) wereadded to tubes 1 and 2; 1 μl of reaction 2 above and 1 μl of MMLV-RTwere added to tubes 3 and 4; 1 μl of reaction 3 above and 1 μl ofMMLV-RT were added to tubes 5 and 6; and 1 μl of reaction 3 was added totubes 7 and 8. The tubes were incubated 20 min at 37° C. and then 2 μlof the solutions were added to 100 μl of L/L and the light output of theresulting mixture was immediately measured using a Turner TD-20eluminometer. The data is presented in Table 49.

A second MM Mix was made for use with T4 DNA Polymerase as follows: 20μl 10×Buffer C (Promega Part #R003A); 5 μl 40 mM sodium pyrophosphate;20 μl 1 μM ADP; 5 μl 1 U/μl NDPK; and 130 μl water. This solution wasmixed by vortex and then used to compose the 8 reaction mixturesdescribed in the paragraph above. Incubations were performed at 37° C.for 20 min and then 2 μl of the reaction mixtures were added to 100 μlof L/L with luciferase. The light output was immediately measured andthe data presented in Table 50 was obtained.

These data show that both of these enzymes can pyrophosphorylate DNAhaving 5′ overhangs. However the T4 DNA polymerase can alsopyrophosphorylate DNA having 3′ overhangs (produced by Sph I digestionof DNA) while the reverse transcriptase cannot utilize this form of DNA.

A final MM Mix was made containing: 20 μl 10×Taq Buffer (Promega Part#M190G); 40 μl 25 mM MgCl₂; 5 μl 40 mM sodium pyrophosphate, and 105 μlof water. This new MM Mix was used to produce 8 mixtures. Mixture 1 and2 contained 17 μl of this new MM Mix, 1 μl of diluted DNA reaction 1,and 2 μl of Taq DNA Polymerase (Promega Part #M166B); mixtures 3 and 4contained 17 μl MM, 1 μl from reaction 2, and 2 μl Taq; mixtures 5 and 6contained 17 μl MM, 1 μl from reaction 3, and 2 μl Taq; and mixture 7and 8 contained 17 μl MM and 1 μl from reaction 3. The mixtures weremixed by vortex action, 30 μl of mineral oil was placed over the tubesand they were incubated at 70° C. for 20 min. Fifteen microliters ofeach tube was removed and 1 μl of 1 U/μl of NDPK and 1.5 μl of 1 μM ADPwas added to each tube. The tubes were incubated at room temperature for15 min, 2.3 μl were removed from each reaction and added to 100 μl ofL/L reagent containing luciferase and the light output of the reactionsmeasured immediately using a luminometer. The data is presented in Table51.

These data show that Taq DNA Polymerase can utilize DNA having a 5′overhang. However, very little light output results when the DNA has a3′ overhang. Thus, Taq polymerase appears to be similar to MMLV-RT inthat it will catalyze the pyrophosphorylation of a DNA if it has a 5′overhang but not if it has a 3′ overhang. T4 DNA Polymerase willcatalyze pyrophosphorylation with either form of DNA overhang. Inaddition, by comparing all the data it is clear that much more light isproduced if the reactions are performed using T4 polymerase than usingeither of the other enzymes.

TABLE 48 Reaction Buffer DNA Bam HI Sph I Water 1 5 μl 1 μl — — 44 μl 25 μl 1 μl 2 μl — 42 μl 3 5 μl 1 μl — 2 μl 42 μl

The solutions used were: Buffer, Promega Buffer B (Part #R002A); DNA,pGEM 3ZF+(1 mg/ml), Promega Part #P227A; Bam HI, Promega Bam HI, Part#R602A, Sph I, Promega Sph I, Part# R626A.

TABLE 49 Reaction Light Units DNA Condition MMLV-RT Added 1 1.472supercoiled + 2 1.445 supercoiled + 3 5.156 linear, 5′ overhang + 44.699 linear, 5′ overhang + 5 1.504 linear, 3′ overhang + 6 1.494linear, 3′ overhang + 7 1.412 linear, 3′ overhang − 8 1.378 linear, 3′overhang −

TABLE 50 Reaction Light Units DNA condition T4 DNAP added 1 2.214supercoiled + 2 1.946 supercoiled + 3 44.46 linear, 5′ overhang + 432.53 linear, 5′ overhang + 5 37.29 linear, 3′ overhang + 6 32.11linear, 3′ overhang + 7 1.446 linear, 3′ overhang − 8 1.361 linear, 3′overhang −

TABLE 51 Reaction Light Units DNA Condition Taq DNAP added 1 1.125supercoiled + 2 1.174 supercoiled + 3 8.110 linear, 5′ overhang + 49.687 linear, 5′ overhang + 5 1.623 linear, 3′ overhang + 6 1.515linear, 3′ overhang + 7 1.004 linear, 3′ overhang − 8 1.046 linear, 3′overhang −

Example 31 Detection of Genomic DNA

In this example, high molecular weight DNA is measured usingpyrophosphorylation of the DNA, transfer of the terminal phosphate fromthe dNTPs to ADP to form ATP and measurement of the ATP usingLuciferase. High molecular weight DNA can be detected at a highersensitivity if it is first cleaved using endonucleases.

The reactions were assembled as in Table 52. The materials used in thereactions were: Buffer; 10×Multicore Buffer (Promega Part #R999A); YeastDNA, S. cerevisiae DNA (380 μg/ml) (Promega Part #G301A); Mouse DNA (300μg/ml) (Promega Part #G309A); Eco RI, Endonuclease Eco RI, 12 u/μl,(Promega Part #R601A). The reactions were heated at 37° C. for 60 minthen at 70° C. for 10 min. At that point, 1 μl of each of thesereactions were diluted to 20 μl by the addition of 19 μl of water.

A solution (MM) was made which contained: 24 μl of 10×Buffer C (PromegaPart #R003A); 6 μl of 40 mM Sodium Pyrophosphate, 24 μl of 1 μM ADP, 6μl of 1 u/μl NDPK, and 156 μl of water. The reactions presented in Table53 were assembled using this mix.

The added DNAs in the reactions 1A through 10A above refer to thediluted materials from reactions 1-4 described in Table 53. The T4 DNAPol is T4 DNA Polymerase (Promega Part #M421F). These reactions wereincubated at 37° C. for 20 min, then 2 μl of the reactions was added to100 μl of L/L reagent. The light produced by the reactions wasimmediately measured using a Turner TD-20e luminometer. The data ispresented in Table 54. Note that the reactions demonstrate that thesystem can detect genomic DNA. In addition, Eco RI treatment prior topyrophosphorylation results in higher light values than are seen withoutEco RI pretreatment.

TABLE 52 Reaction Buffer Yeast DNA Mouse DNA Water Eco RI 1 5 μl 2.6 μl— 42.4 μl — 2 5 μl — 3.3 μl 41.7 μl — 3 5 μl 2.6 μl — 40.4 μl 2 μl 4 5μl — 3.3 μl 39.7 μl 2 μl

TABLE 53 Reaction MM DNA Added T4 DNA Pol 1A and 2A 18 μl 1 μl #1 1 μl3A and 4A 18 μl 1 μl #2 1 μl 5A and 6A 18 μl 1 μl #3 1 μl 7A and 8A 18μl 1 μl #4 1 μl 9A 18 μl 1 μl #3 — 10A  18 μl 1 μl #4 —

TABLE 54 Reaction Light Eco R1 T4 DNA Pol Sampled Units TreatmentTreatment 1A 2.424 − + 2A 1.94 − + 3A 1.989 − + 4A 1.665 − + 5A12.27 + + 6A 11.9 + + 7A 23.23 + + 8A 20.26 + + 9A 0.651 + − 10A 0.724 + −

Example 32 Optimization of ADP Concentrations Used in DNA Detection byPyrophosphorylation.

In this example, we examine the effect of varying the ADP concentrationon the detection of DNA by the T4 DNA Polymerase catalyzedpyrophosphorylation of the DNA and transfer of the terminal phosphatesof the dNTPs to ADP using NDPK. Increasing the concentration of ADPincreases the background seen without ATP addition. Increasing the ADPconcentration also can increase the signal seen upon DNApyrophosphorylation. An optimal amount of added ADP can be determined byselecting the concentration of ADP which results in the best foldincrease in signal over background.

ADP (Sigma potassium ADP, A-5285, Lot #56H7815) was dissolved indistilled water to various concentrations ranging from 0.2 to 20 μM. TheBam HI digest of pGEM 3ZF+described in example 26 above was used to forma reaction solution (solution MM) composed of: 40 μl 10×Buffer C(Promega Part #R003A), 10 μl of 40 mM sodium pyrophosphate; 10 μl of 1u/μl of NDPK; 1 μl of 20 ng/μl Bam HI digested pGEM 3ZF+; and 299 μl ofwater. These solutions were used to compose the reactions presented inTable 55.

As ADP concentration increases, the total light value increases for boththe reactions containing polymerase and those without polymerase asdemonstrated in Table 56. In this example the best fold increase in thesignal, as defined as fold increase in signal over background, is seenwith 0.05 μM ADP in the pyrophosphorylation reaction.

TABLE 55 Reaction RM T4 DNA Pol ADP 1, 2 18 μl 1 μl 1 μl of 0.2 μM 3 18μl — 1 μl of 0.2 μM  4, 5 18 μl 1 μl 1 μl of 1 μ M 6 18 μl —  1 μl of 1μM 7, 8 18 μl 1 μl  1 μl of 2 μM 9 18 μl —  1 μl of 2 μM 10, 11 18 μl 1μl 1 μl of 10 μM 12 18 μl — 1 μl of 10 μM 13, 14 18 μl 1 μl 1 μl of 20μM 15 18 μl — 1 μl of 20 μM The T4 DNA Pol used was Promega T4 DNAPolymerase (10 u/ul) (Pt #M421F)

TABLE 56 Reac- Fold Above tion Light ADP DN Avg. Blank Avg. nopolymerase  1 9.19 0.01 μM + 8.81 8.317 16.9  2 8.43 0.01 μM +  3 0.430.01 μM −  4 28.36 0.05 μM + 29.05 28.39 42.9  5 29.74 0.05 μM +  60.662 0.05 μM −  7 43.29 0.10 μM + 41.54 40.18 29.6  8 39.78 0.10 μM + 9 1.359 0.10 μM − 10 77.4 0.50 μM + 74.9 68.9 11.5 11 72.49 0.50 μM +12 5.969 0.50 μM − 13 82.4  1.0 μM + 80.1 69.38 6.42 14 77.98  1.0 μM +15 10.81  1.0 μM −

Example 33 Detection of ATP Using Fluorescence-Based Methods

In addition to detecting ATP by luciferase-based methods, ATP can bedetected using fluorescence-based systems. For the fluorescence-basedmeasurements, an ATP determination kit was used (Sigma #366-ALot#117H6017). This kit uses a combination of 2 enzymes,phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase, tocatalyze the formation of NAD from NADH in the presence of ATP. Sincethe NADH is fluorescent, but the NAD is not, ATP can be measured as aloss in fluorescence intensity. The reaction buffer was prepared fromkit components as follows: 3 ml of the supplied buffer solution wasdiluted in 5.25 ml of nanopure water, and 0.75 ml of 12% trichloroaceticacid was added. One vial of the supplied NADH was reconstituted in 1 mlof nanopure water; the enzyme mix was used as supplied. For eachmeasurement, 10 μl of enzyme mix and 20 μl of NADH were added to 1.5 mlof reaction buffer in a clear plastic 10 mm cuvette. Fluorescence wasread in a SPEX Fluorolog Fluorimeter using SPEX dm3000 Software, withabsorbance and emission wavelengths set at 340 nm and 460 nm,respectively.

ATP samples at various concentrations were prepared by serially dilutingATP tenfold into 10 mM Tris, pH 7.3. Varying amounts of each dilutionwas added to the cuvette and the decrease in fluorescence was recorded(Table 57). For comparison ATP was also quantitated using luciferase. 20μl of each ATP dilution was added to 100 μl LAR with 10 ng of luciferaseand light output was measured using a TD-20e luminometer. Each dilutionwas measured in duplicate (Table 58).

This example indicates that ATP can be detected by at least two separatemethods. In the fluorescence-based system, changes of approximately200,000 fluorescent light units were significant, which corresponds to 1nanomole of ATP. The luciferase assay was sensitive to lower levels ofATP.

TABLE 57 Decrease in ATP Volume Mass Fluorescence Units in ConcentrationAdded Added 10,000's 10 mM 20 μl 200 nmoles 135 nd^(a) nd^(a) 1 mM 20 μl20 nmoles 84.3 132 nd^(a) 1 mM 10 μl 10 nmoles 89.3 nd^(a) nd^(a) 1 mM 5 μl 5 nmoles 76.4 nd^(a) nd^(a) 100 μM 40 μl 4 nmoles 66.7 60.2 nd^(a)100 μM 20 μl 2 nmoles 23.9 21.9 20.8 100 μM 10 μl 1 nmole 19.1 22.0 18.9100 μM  5 μl 500 pmoles 7.6 6.9 6.8 10 μM 20 μl 200 pmoles 11.6 10.011.1 10 μM 10 μl 100 pmoles 10.4 6.9 6.6 1 μM 20 μl 20 pmoles 8.2 8.45.2 1 μM 10 μl 10 pmoles 8.0 8.1 5.3 0.1 μM 20 μl 2 pmoles 3.2 5.6 3.60.01 μM 20 μl 200 fmoles 8.1 9.7 6.8 Tris 20 μl — 4.3 3.7 3.8 Tris 10 μl— 4.0 3.3 3.5 nd^(a),not done

TABLE 58 APT, 20 μl of Light Units 10 mM 102,417 102,731 1 mM 117,71898,842 100 μM 47,676 44,101 10 μM 7690 6998 1 μM 812 798 0.1 μM 76.867.8 0.01 μM 7.0 4.5 Tris 0.06 0.06

Example 34 Detection of ATP Using Fluorescence; PRPP Synthetase,Reactions with Adenosine

ATP was synthesized by the enzyme PRPP Synthetase from the substratesAMP and PRPP as in Example 13, except the reactions were done in largervolumes and the substrates were at higher concentrations. 20 μl of AMP(29 mM) and 20 μl of PRPP (26 mM) were incubated with 20 μl of PRPPSynthetase (6×10⁻³ units) in 200 μl of PRPP Synthetase buffer. Thereactions are summarized in Table 59. After a 30 minute incubation at37° C., the PRPP Synthetase was heat-inactivated at 94° C. for 10minutes. The ATP was then quantitated using both a fluorescence-basedsystem and a luciferase-based system. For the fluorescence-basedmeasurements, an ATP determination kit was used (Sigma #366-ALot#117H6017) as described in Example 33. Twenty microliter aliquots ofthe PRPP reactions were then added to cuvettes containing 1.5 ml ofbuffer, 10 μl of enzyme mix and 20 μl of NADH. The decrease influorescence was monitored. Four to six measurements were made for eachreaction (Table 60). For the luciferase-based assay, 20 μl was added to100 μl of LAR and 10 ng of luciferase. Each reaction was determined intriplicate. Light output was measured using a Turner TD-20e luminometer(Table 61). This example demonstrates that ATP production by PRPPSynthetase can be measured using fluorescence or luciferase.

TABLE 59 PRPP Syn PRPP Reaction Buffer AMP PRPP Synthetase 1 200 μl 20μl 20 μl 20 μl 2 200 μl 20 μl — 20 μl 3 200 μl — 20 μl 20 μl 4 200 μl 20μl 20 μl —

TABLE 60 Decrease in Fluorescence Units Reaction (in 10,000's) Average 149.1 48.0 47.3 49.0 nd^(a) nd^(a) 48.4 2 2.48 3.30 2.37 10.9 7.06 9.575.95 3 3.36 2.30 11.06 7.63 10.5 nd^(a) 6.97 4 3.48 1.68 4.83 0.62 5.743.37 3.29 nd^(a),not done

TABLE 61 Reaction Light Units 1 8923 9995 9562 2 0.001 0.000 0.013 31939 17G0 1770 4 27.9 23.7 23.0

Example 35 Detection of ATP Using Fluorescence; Cell Lysates

ATP can also be generated by incubating cell lysates with AMP and dCTPas described in Examples 25, 26 and 27. The Sigma ATP determination kitdescribed in Example 33 was also used to detect ATP in this system.Reactions were assembled as described below (Table 62) and incubated atroom temperature. ATP concentrations were quantitated at 80 minutes and140 minutes using luciferase. In this assay 15 μl of each reaction wasadded to 100 μl of LAR and 10 ng of luciferase. Light output wasmeasured using a Turner Luminometer TD-20e (Table 63). During the timecourse, ATP was also measured by fluorescence. The procedure was asdescribed in Example 33, except that 15 μl of each reaction was addedper reading, instead of 20 μl. The first set of time points began at 80minutes; the second set of readings began at 140 minutes. Each reactionwas assayed in duplicate or triplicate (Table 64). This exampledemonstrates that ATP synthesized in cell lysates can be detected usinga luciferase or a fluorescence assay.

TABLE 62 E. coli 0.05 M 100 mM Reaction Lysate MgSO₄ 10 mM AMP dCTP 1100 μl 20 μl 20 μl 10.5 μl 2 — 20 μl 20 μl 10.5 μl 3 100 μl — 20 μl 10.5μl 4 100 μl 20 μl — 10.5 μl 5 100 μl — — 10.5 μl

TABLE 63 Light Units Reactions T = 80 minutes T = 140 minutes 1 33,51965,522 2 2.158 2.086 3 362.7 370.6 4 0.5 0.561 5 1.898 1.057

TABLE 64 Decrease in Fluorescence Units (in 10,000's) Reaction FirstTime Point Second Time Point 1 27.1 29.4 83.8 87.3 2 11.9 8.2 1.3 1.2 312.2 8.2 4.1 4.7 4 5.0 4.1 4.2 2.8 5 nd^(a) nd^(a) 4.8 7.3 nd^(a),notdone

Example 36 Extremely Sensitive DNA Measurement by Amplification ofPyrophosphorylation Reaction Products

This Example demonstrates that AMP can be a source of extraneousnucleotides that result in unwanted background amplification inreactions spiked with a nucleoside triphosphate and that the detectionlimit for DNA measured through the pyrophosphorylation of the sample canbe lowered if the products are amplified.

Two reactions were assembled. Reaction 1 consisted of: 2 μl of 10×BufferC (Promega Corp. R003A, Lot 7544205); 0.5 μl of 40 mM sodiumpyrophosphate; 2 μl of 1 mM AMP; 1 ul of 0.25 U/μl Myokinase (SigmaM3003, Lot 116H9516); 1 μl of 0.17 U/ul Pyruvate Kinase (Sigma N 0379,Lot 127F81802); 1 μl of 10 U/μl T4 DNA Polymerase (Promega M421F Lot617506) and 11.5 μl of water. Reaction 2 was identical to Reaction 1except that the AMP was treated with Apyrase in a reaction consisting of20 μl of 10 mM AMP and 1 μl of 1 U/μl Apyrase (Sigma A 6535 lot127H7010) for 30 min at room temperature, followed by a heatinactivation step to eliminate the Apyrase activity by treatment at 70°C. for 10 Min.

At time 0, 1 μl of 10 mM PEP was added to each reaction and the reactionwas mixed and incubated at room temperature. At 2 min, 2 μl of thereaction was removed and added to 100 μl of L/L reagent and the lightoutput measured using a Turner TD-20e Luminometer as described above.The following data were collected: Reaction 1; 817.4 light units,Reaction 2; 7.3 light units. Since there should be no ATP produced bythis reaction unless extraneous nucleoside di- or triphosphate is addedas a contaminant in a reagent, this demonstrates that the AMP probablycontained some level of contaminating nucleotide which was eliminated byApyrase treatment.

The following reactions were assembled: Reaction A contained thecomponents described in Reaction 2 above except that 1 μl of 1 ng Hin F1Fragments(Promega Corp, G175A Lot 7733602) diluted to this concentrationwith 1×TE Buffer )/μl was added and the T4 DNA Polymerase was not addedto the initial reaction mix; Reaction B, same as Reaction A but the DNAadded was at a concentration of 100 pg DNA/μl; Reaction C, same asReaction A but the DNA added was at a concentration of 10 pg DNA/μl;Reaction D, same as Reaction A but the DNA added was at a concentrationof 1 pg DNA/μl; and, Reaction E, same as Reaction A but with 1 μl of1×TE Buffer added and no DNA added.

One microliter of T4 DNA Polymerase was added to each reaction and thereactions were incubated at 37° C. for 15 min. After this incubation, 1μl of 10 mM PEP was added to each reaction and incubated again at roomtemperature for 10 min. At that time, 2 μl of each reaction was added to100 μl of L/L reagent and the light output of the reaction was measuredusing a Turner Luminometer as described above. The data are presented inTable 65. This Example demonstrates that the products of thepyrophosphorylation reaction can be coupled to an ATP amplificationsystem to increase the sensitivity of DNA measurement.

TABLE 65 Light Measured From Samples Incubated at Room Temperature pgDNA* 10 min 20 min A 100 pg 917.3 1156  B 10 pg 112.1 1119  C 1 pg 4.68919 D 0.1 pg 2.61 873 E 0 1.52 650 *The DNA reported in this column isthe actual DNA equivalent Luciferase reaction. The amount isapproximately 10% of the total pyrophosphorylated.

What is claimed is:
 1. A method of detecting deoxyribonucleic acid in areaction containing pyrophosphate, adenosine 5′-diphosphate, or acombination thereof, the method comprising: depolymerizing adeoxyribonucleic acid at a terminal nucleotide by enzymatically cleavingthe terminal internucleotide phosphodiester bond and reforming same witha pyrophosphate molecule to form a free deoxyribonucleoside triphosphatemolecule according to the reaction: dNA_(n)+PP_(i)→dNA_(n−1)+dNTP;enzymatically transferring terminal 5′ phosphate groups from thedeoxyribonucleoside triphosphate molecules to adenosine 5′-diphosphatemolecules to form adenosine 5′-triphosphate according to the followinggeneral reaction: dNTP*+ADP→dNDP+ATP*, wherein P* is the terminal 5′phosphate so transferred; and detecting the adenosine 5′-triphosphateformed thereby.
 2. The method of claim 1 wherein the depolymerizing stepis catalyzed by a DNA polymerase or reverse transcriptase selected fromthe group consisting of T4 polymerase, Taq polymerase, AMV reversetranscriptase and MMLV reverse transcriptase.
 3. The method of claim 1,wherein the step of enzymatically transferring terminal 5′ phosphategroups from the nucleoside triphosphate molecules to adenosine5′-diphosphate molecules to form adenosine 5′-triphosphate molecules iscatalyzed by nucleoside diphosphate kinase.
 4. The method of claim 1,wherein the ATP detection method is a luciferase detection system. 5.The method of claim 1, wherein the ATP detection method is a NADHdetection system.
 6. The method of claim 1, wherein the depolymerizingstep and phosphate transferring steps are performed in a single potreaction.
 7. The method of claim 1, together with the step of amplifyingthe adenosine 5′-triphosphate molecules produced by the phosphatetransferring step.
 8. The method of claim 7 wherein the amplification isperformed according to the method of claim
 1. 9. A method of producing aplurality of adenosine triphosphate molecules from a nucleosidetriphosphate molecule in a reaction containing adenosine5′-monophosphate molecules, high energy phosphate donor molecules, or acombination thereof, the method comprising the steps of: (1)enzymatically transferring the terminal 5′ phosphate group from anucleoside triphosphate molecule present in the sample to an adenosine5′-monophosphate molecule added to the sample to form an adenosine5′-diphosphate molecule and nucleoside diphosphate molecule according tothe following general reaction catalyzed by a first enzyme:XTP+AMP→XDP+ADP; (2) enzymatically transferring a phosphate group from ahigh energy phosphate donor molecule which may not be utilized by thefirst enzyme to adenosine 5′-diphosphate molecules to form adenosine5′-triphosphate molecules according to the following general reactioncatalyzed by nucleoside diphosphate kinase: ADP+D−P=ATP+D; andamplifying the adenosine triphosphate molecule so produced by repeatingsteps (1) and (2) until the desired level of amplification is achieved.10. A method of detecting polyadenylated mRNA in a reaction containingpyrophosphate, the method comprising: depolymerizing the polyadenylatedmRNA at a terminal nucleotide by enzymatically cleaving the terminalinternucleotide phosphodiester bond and reforming same with apyrophosphate molecule to form a free adenosine triphosphate moleculeaccording to the reaction catalyzed by poly(A) polymerase:NA_(n)+PP_(i)→NA_(n−1)+ATP; and detecting the adenosine 5′-triphosphateformed thereby.
 11. The method of claim 10, wherein the ATP detectionmethod is a luciferase detection system.
 12. The method of claim 10,wherein the ATP detection method is a NADH detection system.
 13. Themethod of claim 10, together with the step of amplifying the adenosine5′-triphosphate molecules produced by the depolymerizing step.
 14. Themethod of claim 13 wherein the amplification is performed according tothe method of claims
 1. 15. A method of selectively detectingpoly(A)-mRNA in a reaction containing pyrophosphate, adenosine5′-diphosphate, or a combination thereof, the method comprising:hybridizing a complimentary oligo(dT) probe to poly(A)-mRNA to form aRNA-DNA hybrid, depolymerizing the oligo(dT) strand of the RNA-DNAhybrid at the terminal nucleotide by enzymatically cleaving the terminalinternucleotide phosphodiester bond and reforming same with apyrophosphate molecule to form a deoxythymidine 5′-triphosphate moleculeaccording to the following general reaction catalyzed by a reversetranscriptase dTT_(n)+PP_(i)→dTT_(n−1)+dTTP; enzymatically transferringterminal 5′ phosphate groups from the deoxythymidine 5′-triphosphatemolecules to adenosine 5′-diphosphate molecules to form adenosine5′-triphosphate molecules according to the following general reaction:dTTP*+ADP=dTDP+ATP*, wherein P* is the terminal 5′ phosphate sotransferred; and detecting the adenosine 5′-triphosphate formed thereby.16. The method of claim 15, wherein the ATP detection method is aluciferase detection system.
 17. The method of claim 15, wherein the ATPdetection method is a NADH detection system.
 18. The method of claim 15,together with the step of amplifying the adenosine 5′-triphosphatemolecules produced by the depolymerizing step.
 19. The method of claim18 wherein the amplification is performed according to the method ofclaim
 1. 20. A method of detecting DNA in a reaction containingphosphoribosylpyrophosphate, adenosine 5′-diphosphate, or a combinationthereof, the method comprising: producing free deoxyadenosinemonophosphate molecules from the nucleic acid by digestion with anuclease; enzymatically transferring the pyrophosphate fromphosphoribosylpyrophosphate molecules to the deoxyadenosinemonophosphate molecules to form deoxyadenosine triphosphate moleculesaccording to the following reaction catalyzed byphosphoribosylpyrophosphate synthetase dAMP+PRPP→dATP+ribose-5-PO₄;enzymatically transferring terminal 5′ phosphate groups from thedeoxyribonucleoside triphosphate molecules to adenosine 5′-diphosphatemolecules to form adenosine 5′-triphosphate molecules according to thefollowing general reaction: dATP*+ADP→dADP+ATP*, wherein P* is theterminal 5′ phosphate so transferred; and detecting the ATP so produced.21. The method of claim 20, wherein the enzymatic transfer of phosphatefrom the free deoxyadenosine triphosphate molecules to adenosine5′-diphosphate molecules is catalyzed by nucleoside diphosphate kinase.22. The method of claim 20, wherein the detection system is a luciferasedetection system.
 23. The method of claim 20, wherein the detectionsystem is a NADH detection system.
 24. The method of claim 20, whereinthe pyrophosphate transferring step and the phosphate transferring stepare performed in a single pot reaction.
 25. The method of claim 20,together with the step of amplifying the adenosine 5′-triphosphatemolecules produced by the phosphate transferring step.
 26. The method ofclaim 25 wherein the amplification is performed according to the methodof claim
 1. 27. A method of detecting RNA in a reaction containingphosphoribosylpyrophosphate, the method comprising: producing freeribonucleoside monophosphate molecules from the RNA by digestion with anuclease; enzymatically transferring the pyrophosphate fromphosphoribosylpyrophosphate molecules to the adenosine monophosphatemolecules to form adenosine triphosphate molecules according to thefollowing reaction catalyzed by phosphoribosylpyrophosphate synthetaseAMP+PRPP→ATP+ribose-5-PO₄; and detecting the ATP so produced.
 28. Themethod of claim 27, wherein the detection system is a luciferasedetection system.
 29. The method of claim 27, wherein the detectionsystem is a NADH detection system.
 30. The method of claim 27, togetherwith the step of amplifying the adenosine 5′-triphosphate moleculesproduced by the phosphate transferring step.
 31. The method of claim 30wherein the amplification is performed according to the method ofclaim
 1. 32. A kit containing reagents for the detection of DNA bypyrophosphorolysis comprising: a vessel containing a nucleic acidpolymerase; and a vessel containing a nucleoside diphosphate kinase. 33.The kit of claim 32 wherein the nucleic acid polymerase and nucleosidediphosphate kinase are provided in the same container.
 34. A kitcontaining reagents for the detection of DNA by nuclease digestioncomprising: a vessel containing phosphoribosylpyrophosphate synthetase;and a vessel containing a nucleoside diphosphate kinase.
 35. The kit ofclaim 34 wherein the phosphoribosylpyrophosphate synthetase andnucleoside diphosphate kinase are provided in the same container.
 36. Amethod of detecting deoxyribonucleic or ribonucleic acid in a reactioncontaining pyrophosphate, adenosine 5′-monophosphate and a high energyphosphate donor, or a combination thereof, the method comprising thefollowing steps performed in a single pot reaction: depolymerizing anucleic acid at a terminal nucleoside by enzymatically cleaving theterminal internucleotide phosphodiester bond and reforming same with apyrophosphate molecule to form a free ribonucleotide ordeoxyribonucleoside triphosphate molecule according to reaction 1:NA_(n)+PP_(i)→NA_(n−1)+XTP; repeating the depolymerizing step to obtainat least two nucleoside triphosphate molecules; producing a plurality ofadenosine triphosphate molecules from the ribonucleoside triphosphate ordeoxyribonucleoside phosphate molecules by: (i) enzymaticallytransferring the terminal 5′ phosphate group from the ribonucleoside ordeoxyribonucleoside triphosphate molecule formed in reaction 1 to anadenosine 5′-monophosphate molecule to produce an adenosine5′-diphosphate molecule and a ribonucleoside or deoxyribonucleosidediphosphate molecule according to reaction 2 catalyzed by a firstenzyme: XTP+AMP=XDP+ADP; (ii) enzymatically transferring a phosphategroup from a high energy phosphate donor molecule which is not asubstrate for the first enzyme to the adenosine 5′-diphosphate moleculesproduced in reaction 2 to produce an adenosine 5′-triphosphate moleculesaccording to reaction 3 catalyzed by a second enzyme: ADP+D-P=ATP+D; and(iii) amplifying the adenosine triphosphate molecules so produced byrepeating steps (i) and (ii) until the desired level of amplification isachieved; and detecting the ATP so produced.
 37. The method of claim 36wherein said first enzyme is selected from the group consisting ofadenylate kinase and nucleoside monophosphate kinase.
 38. The method ofclaim 36 where said second enzyme is selected from the group consistingof pyruvate kinase and nucleoside diphosphate kinase.
 39. A kitcontaining reagents for the detection of nucleic acid by nucleasedigestion comprising: a vessel containing phosphoribosylpyrophosphatesynthetase; and a vessel containing a nuclease.
 40. A kit containingreagents for the detection of RNA by pyrophosphorolysis comprising: avessel containing poly(A)-polymerase.
 41. A kit containing reagents forthe detection of cells and/or cellular material in a sample comprising:a vessel containing adenosine 5′-monophosphate; and a vessel containinga high energy phosphate donor which may not be utilized by luciferase.42. A method of assaying deoxyribonucleic acid in a reaction containingpyrophosphate, adenosine 5′-diphosphate, or a combination thereof, themethod comprising: depolymerizing a nucleic acid at a terminalnucleotide by enzymatically cleaving the terminal internucleotidephosphodiester bond and reforming same with a pyrophosphate molecule toform a free deoxyribonucleoside triphosphate molecule according to thereaction: dNA_(n)+PP_(i)→dNA_(n−1)+dNTP; repeating the depolymerizingstep to obtain at least two deoxyribonucleoside triphosphate molecules;enzymatically transferring terminal 5′ phosphate groups from thedeoxyribonucleoside triphosphate molecules to adenosine 5′-diphosphatemolecules to form adenosine 5′-triphosphate according to the followinggeneral reaction: dNTP*+ADP→dNDP+ATP*, wherein P* is the terminal 5′phosphate so transferred; and detecting the adenosine 5′-triphosphateformed thereby.
 43. The method of claim 42 wherein the depolymerizingstep is catalyzed by a DNA polymerase or reverse transcriptase selectedfrom the group consisting of T4 polymerase, Taq polymerase, AMV reversetranscriptase and MMLV reverse transcriptase.
 44. The method of claim42, wherein the step of enzymatically transferring terminal 5′ phosphategroups from the nucleoside triphosphate molecules to adenosine5′-diphosphate molecules to form adenosine 5′-triphosphate molecules iscatalyzed by nucleoside diphosphate kinase.
 45. The method of claim 42,wherein the ATP detection method is a luciferase detection system. 46.The method of claim 42, wherein the ATP detection method is a NADHdetection system.
 47. The method of claim 42, wherein the depolymerizingstep and phosphate transferring steps are performed in a single potreaction.
 48. The method of claim 42, together with the step ofamplifying the adenosine 5′-triphosphate molecules produced by thephosphate transferring step.
 49. The method of claim 48 wherein theamplification is performed according to the method of claim
 9. 50. Amethod of assaying polyadenylated mRNA in a reaction containingpyrophosphate, the method comprising: depolymerizing the polyadenylatedmRNA at a terminal nucleotide by enzymatically cleaving the terminalinternucleotide phosphodiester bond and reforming same with apyrophosphate molecule to form a free adenosine triphosphate moleculeaccording to the reaction catalyzed by poly(A) polymerase:NA_(n)+PP_(i)→NA_(n−1)+ATP; repeating the depolymerizing step to obtainat least 2 nucleoside triphosphate molecules; and detecting theadenosine 5′-triphosphate formed thereby.
 51. The method of claim 50,wherein the ATP detection method is a luciferase detection system. 52.The method of claim 50, wherein the ATP detection method is a NADHdetection system.
 53. The method of claim 50, together with the step ofamplifying the adenosine 5′-triphosphate molecules produced by thedepolymerizing step.
 54. The method of claim 53 wherein theamplification is performed according to the method of claims
 9. 55. Amethod of selectively assaying poly(A)-mRNA in a reaction containingpyrophosphate, adenosine 5′-diphosphate, or a combination thereof, themethod comprising: hybridizing a complimentary oligo(dT) probe topoly(A)-mRNA to form a RNA-DNA hybrid, depolymerizing the oligo(dT)strand of the RNA-DNA hybrid at the terminal nucleotide by enzymaticallycleaving the terminal internucleotide phosphodiester bond and reformingsame with a pyrophosphate molecule to form a deoxythymidine5′-triphosphate molecule according to the following general reactioncatalyzed by a reverse transcriptase dTT_(n)+PP_(i)→dTT_(n−1)+dTTP;repeating the depolymerizing step to obtain at least two nucleosidetriphosphate molecules; enzymatically transferring terminal 5′ phosphategroups from the deoxythymidine 5′-triphosphate molecules to adenosine5′-diphosphate molecules to form adenosine 5′-triphosphate moleculesaccording to the following general reaction: dTTP*+ADP=dTDP+ATP*,wherein P* is the terminal 5′ phosphate so transferred; and detectingthe adenosine 5′-triphosphate formed thereby.
 56. The method of claim55, wherein the ATP detection method is a luciferase detection system.57. The method of claim 55, wherein the ATP detection method is a NADHdetection system.
 58. The method of claim 55, together with the step ofamplifying the adenosine 5′-triphosphate molecules produced by thedepolymerizing step.
 59. The method of claim 58 wherein theamplification is performed according to the method of claim 9.